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Proteome of monoclonal antibody-purified haustoria from Puccinia triticina Race-1
Abstract
Puccinia triticina causes leaf rust, a disease that causes annual yield losses on wheat. It is an obligate parasite that invades the host leaf and forms intracellular structures called haustoria, which obtain nutrients and suppress host immunity using secreted proteins called effectors. Since effector proteins act at the frontier between plant and pathogen and help determine the outcome of the interaction, it is critical to understand their functions. Here we used a direct proteomics approach to identify effector candidates from P. triticina Race 1 haustoria isolated with a specific monoclonal antibody. Haustoria were >95% pure and free of host contaminants. Using high resolution mass spectrometry we have identified 1192 haustoria proteins. These were quantified using normalized spectral counts and spanned a dynamic range of 3 orders of magnitude, with unknown proteins and metabolic enzymes as the most highly represented. A survey of post-translational modification indicated that the most abundant were oxidative modifications. The dataset contained 140 candidate effector proteins, based on the presence of a signal peptide and the absence of a known function for the protein. Some of these candidates were significantly enriched with cysteine, with up to 13 residues per protein and up to 6.8% cysteine in composition.This article is protected by copyright. All rights reserved
... Of these orthologs, the AvrLr2a candidate GN104ID162_007386, the AvrLr3ka candidate GN104ID162_024924, and the AvrLr26 candidate GN104ID162_020918 had corresponding orthologs of PTTG_07365, PTTG_28070, and PTTG_11943 in the race1 genome, respectively (Supplementary Table S6). In agreement with these findings, these orthologs from Pt race1 were also predicted as candidate effectors based on a proteomics study of haustoria isolated from Pt race1 (Rampitsch et al., 2015). ...
... The other study was for the American Pt BBD race1, which used various DNA libraries (e.g., fosmid and BAC libraries) and sequencing platforms (e.g., Roche 454 and Sanger sequencing) to build an assembly comprising 14,818 scaffolds with an N 50 length of 544 kb (Cuomo et al., 2016). Although this assembly has better quality and has been used as a reference genome by a couple of transcriptome and proteome studies as well as our study on AvrLr20 identification (Song et al., 2011;Bruce et al., 2014;Rampitsch et al., 2015;Wu et al., 2017), the major issue of high fragmentation largely due to the limitation of short-read sequencing and repetitive nature of rust genomes remains to be resolved (Aime et al., 2017). While our LRS-based Pt104 assembly has a genome length close to 135.3 Mb as previously reported for Pt race1, our assembly is significantly improved in terms of contiguity and completeness as exemplified by 91-fold reduction in the number of contigs, 4-fold improvement in N 50 statistics (contigs N 50 versus scaffolds N 50 ; Figure 1 and Table 1), and no missing data represented by Ns (Cuomo et al., 2016). ...
... Differential variants derived from the pairwise comparisons set up with contrasting virulence profiles (Supplementary Tables S3, S4) led to the identification of 38, 31, and 37 Avr genes as candidates for AvrLr26, AvrLr2a, and AvrLr3ka, respectively (Figure 5 and Supplementary Table S6). Interestingly, three of the candidate genes had orthologs in Pt race1 as aforementioned, which were also predicted as potential effectors in a proteomic study of haustoria isolated from race1 (Rampitsch et al., 2015). This consistency provided further support for our candidate genes at the level of haustorial proteomes. ...
Leaf rust, caused by Puccinia triticina (Pt), is one of the most devastating diseases of wheat, affecting production in nearly all wheat-growing regions worldwide. Despite its economic importance, genomic resources for Pt are very limited. In the present study, we have used long-read sequencing (LRS) and the pipeline of FALCON and FALCON-Unzip (v4.1.0) to carry out the first LRS-based de novo genome assembly for Pt. Using 22.4-Gb data with an average read length of 11.6 kb and average coverage of 150-fold, we generated a genome assembly for Pt104 [strain 104-2,3,(6),(7),11; isolate S423], considered to be the founding isolate of a clonal lineage of Pt in Australia. The Pt104 genome contains 162 contigs with a total length of 140.5 Mb and N50 of 2 Mb, with the associated haplotigs providing haplotype information for 91% of the genome. This represents the best quality of Pt genome assembly to date, which reduces the contig number by 91-fold and improves the N50 by 4-fold as compared to the previous Pt race1 assembly. An annotation pipeline that combined multiple lines of evidence including the transcriptome assemblies derived from RNA-Seq, previously identified expressed sequence tags and Pt race 1 protein sequences predicted 29,043 genes for Pt104 genome. Based on the presence of a signal peptide, no transmembrane segment, and no target location to mitochondria, 2,178 genes were identified as secreted proteins (SPs). Whole-genome sequencing (Illumina paired-end) was performed for Pt104 and six additional strains with differential virulence profile on the wheat leaf rust resistance genes Lr26, Lr2a, and Lr3ka. To identify candidates for the corresponding avirulence genes AvrLr26, AvrLr2a, and AvrLr3ka, genetic variation within each strain was first identified by mapping to the Pt104 genome. Variants within predicted SP genes between the strains were then correlated to the virulence profiles, identifying 38, 31, and 37 candidates for AvrLr26, AvrLr2a, and AvrLr3ka, respectively. The identification of these candidate genes lays a good foundation for future studies on isolating these avirulence genes, investigating the molecular mechanisms underlying host–pathogen interactions, and the development of new diagnostic tools for pathogen monitoring.
... The control of leaf rust resistance has also been shown to have a component of epigenetics, involving noncoding RNAs (ncRNAs), DNA/RNA methylation, histone modifications, and chromatin remodeling (Ramirez-Prado et al., 2018). For instance, differential methylation was observed between two pairs of wheat nearisogenic lines (NILs) namely TcLr19/Thatcher and TcLr41/Thatcher, when DNA methylation was examined using methylation-sensitive amplified polymorphism (MSAP) analysis during leaf rust infection (Fu et al., 2009). ...
... In contrast to this two Pgt effectors [AvrSr50 (132 aa) and AvrSr35 (578 aa)], were recently characterized using new generation sequencing and RNA-seq approaches (Chen et al., 2017;Salcedo et al., 2017). Proteome analysis has also been intended for the prediction of candidate effectors (Rampitsch et al., 2015;Song et al., 2011). Some of the advantages of investigating the proteome are that the protein loads are measured directly-eliminating the need to correlate transcript and protein levels as is the case with transcriptome analysis and that most of the post-translational modifications can be identified by mass spectrometry in proteome analysis (Rampitsch et al., 2015). ...
... Proteome analysis has also been intended for the prediction of candidate effectors (Rampitsch et al., 2015;Song et al., 2011). Some of the advantages of investigating the proteome are that the protein loads are measured directly-eliminating the need to correlate transcript and protein levels as is the case with transcriptome analysis and that most of the post-translational modifications can be identified by mass spectrometry in proteome analysis (Rampitsch et al., 2015). Six effector protein signatures (glucan 1,3 b-glucosidase and chitinases, peptidase and proteases, carboxypeptidase, Subtilisin-like serine proteases, Protein disulfide isomerases, and Cyclophilins) were identified in Pt using proteome analysis (Song et al., 2011). ...
Leaf rust (also called brown rust) in wheat, caused by fungal pathogen Puccinia triticina Erikss. (Pt) is one of the major constraints in wheat production worldwide. Pt is widespread with diverse population structure and undergoes rapid evolution to produce new virulent races against resistant cultivars that are regularly developed to provide resistance against the prevailing race of the pathogen. Occasionally, the disease may also take the shape of an epidemic in some wheat-growing areas causing major economic losses. In the recent past, substantial progress has been made in characterizing the sources of leaf rust resistance including non-host resistance (NHR). Progress has also been made in elucidating the population biology of Pt and the mechanisms of wheat-Pt interaction. So far, ∼80 leaf rust resistance genes (Lr genes) have been identified and characterized; some of them have also been used for the development of resistant wheat cultivars. It has also been shown that a gene-for-gene relationship exists between individual wheat Lr genes and the corresponding Pt Avr genes so that no Lr gene can provide resistance unless the prevailing race of the pathogen carries the corresponding Avr gene. Several Lr genes have also been cloned and their products characterized, although no Avr gene corresponding a specific Lr gene has so far been identified. However, several candidate effectors for Pt have been identified and functionally characterized using genome-wide analyses, transcriptomics, RNA sequencing, bimolecular fluorescence complementation (BiFC), virus-induced gene silencing (VIGS), transient expression and other approaches. This review summarizes available information on different aspects of the pathogen Pt, genetics/genomics of leaf rust resistance in wheat including cloning and characterization of Lr genes and epigenetic regulation of disease resistance.
... Table 2 lists CSEPs found in this study up to 72 h post-inoculation. Only one of these was also reported previously from rust haustoria (Rampitsch et al., 2015). Just two CSEPs were detected in the 24 and 48 h harvests; however, it is unclear whether CSEP abundance is often too low for detection. ...
... Just two CSEPs were detected in the 24 and 48 h harvests; however, it is unclear whether CSEP abundance is often too low for detection. Later (5 d) CSEPs likely originate from haustoria and were discussed previously (Song et al., 2011;Rampitsch et al., 2015). ...
... Haustorial mother cells start to form as soon as 24 h after inoculation; however, at this time, there is still an increase in the numbers of appressoria, a much earlier infection structure (Hu and Rijkenberg, 1998). From the present work, it seems clear that haustoria are the main source of secreted proteins as previously reported by Vögele and Mengden (2003) who measured transcript levels, and by this group (Rampitsch et al., 2015), who examined the proteome of purified rust haustoria. The increase of proteins with significantly altered abundance 5 days post-inoculation can be explained by the increased numbers of haustoria at this time point, especially as this increase was seen only in the incompatible interaction between P. triticina-race 1 and Thatcher wheat-the compatible interaction between P. triticina race1 and ThatcherLr1 leads to hypersensitive cell death before haustoria can form (Cloutier et al., 2007 and references therein]. ...
Wheat leaf rust caused by the pathogenic fungus, Puccinia triticina, is a serious threat to bread wheat and durum production in many areas of the world. This plant-pathogen interaction has been studied extensively at the molecular genetics level however, proteomics data are still relatively scarce. The present study investigated temporal changes in the abundance of the apoplastic fluid proteome of resistant and susceptible wheat leaves infected with P. triticina race-1, using a label-free LC-MS-based approach. In general, there was very little difference between inoculated and control apoplastic proteomes in either host, until haustoria had become well established in the susceptible host, although the resistant host responds to pathogen challenge sooner. In the earlier samplings (up to 72 h after inoculation) there were just 46 host proteins with significantly changing abundance, and pathogen proteins were detected only rarely and not reproducibly. This is consistent with the biotrophic lifestyle of P. triticina, where the invading pathogen initially causes little tissue damage or host cell death, which occur only later during the infection cycle. The majority of the host proteins with altered abundance up to 72 h post-inoculation were pathogen-response-related, including peroxidases, chitinases, β-1-3-endo-glucanases, and other PR proteins. Five days after inoculation with the susceptible apoplasm it was possible to detect 150 P. triticina proteins and 117 host proteins which had significantly increased in abundance as well as 33 host proteins which had significantly decreased in abundance. The latter represents potential targets of pathogen effectors and included enzymes which could damage the invader. The pathogen-expressed proteins—seen most abundantly in the incompatible interaction—were mostly uncharacterized proteins however, many of their functions could be inferred through homology-matching with pBLAST. Pathogen proteins also included several candidate effector proteins, some novel, and some which have been reported previously. All MS data have been deposited in the PRIDE archive (www.ebi.ac.uk/pride/archive/) under Project PXD012586.
... Therefore, nanoparticle-mediated delivery of biomolecules in crop plants could be explored for functional characterizations of pathogen effectors. Like the genome and transcriptome analyses, proteome analysis has also been used for the identification of candidate effectors (Song et al. 2011;Rampitsch et al. 2015). One of the most significant advantages of investigating the proteome over transcriptome is that the protein loads are measured directly by removing the need to compare transcript and protein levels. ...
... One of the most significant advantages of investigating the proteome over transcriptome is that the protein loads are measured directly by removing the need to compare transcript and protein levels. In proteome analysis, most post-translational modifications (PTMs) can be identified using mass spectrometry (Rampitsch et al. 2015). Six effector protein signatures (glucan 1,3 b-glucosidase and chitinases, peptidase and proteases, carboxypeptidase, subtilisin-like serine proteases, protein disulfide isomerases and cyclophilins) were identified in Pt using proteome analysis (Song et al. 2011). ...
... Six effector protein signatures (glucan 1,3 b-glucosidase and chitinases, peptidase and proteases, carboxypeptidase, subtilisin-like serine proteases, protein disulfide isomerases and cyclophilins) were identified in Pt using proteome analysis (Song et al. 2011). Rampitsch et al. (2015) also identified six potential candidate effector proteins enriched in cysteine residues in Pt using proteome analysis of monoclonal antibody-purified haustoria from Pt Race-1, five of which (PTTG_05834, PTTG_06324, PTTG_08248, PTTG_06270 and PTTG_03653) were previously described by Song et al. (2011). Moreover, a yeastbased high-throughput screening system, which comprises a GatewayTM-compatible Tet-Off inducible expression vector and a yeast strain expressing a reporter, has been developed for functional characterization of pathogen effectors. ...
Main conclusion
Identification and functional characterization of plant pathogen effectors promise to ameliorate future research and develop effective and sustainable strategies for controlling or containing crop diseases.
Abstact
Wheat is the second most important food crop of the world after rice. Rust pathogens, one of the major biotic stresses in wheat production, are capable of threatening the world food security. Understanding the molecular basis of plant–pathogen interactions is essential for devising novel strategies for resistance breeding and disease management. Now, it has been established that effectors, the proteins secreted by pathogens, play a key role in plant–pathogen interactions. Therefore, effector biology has emerged as one of the most important research fields in plant biology. Recent advances in genomics and bioinformatics have allowed identification of a large repertoire of candidate effectors, while the evolving high-throughput tools have continued to assist in their functional characterization. The repertoires of effectors have become an important resource for better understanding of effector biology of pathosystems and resistance breeding of crop plants. In recent years, a significant progress has been made in the field of rust effector biology. This review describes the recent advances in effector biology of obligate fungal pathogens, identification and functional analysis of wheat rust pathogens effectors and the potential applications of effectors in molecular plant biology and rust resistance breeding in wheat.
... In the present study, we report the genome re-sequencing of 20 Australian Pt isolates, comprising 10 pairs of isolates differing in avirulence/virulence only to Lr20. The reads of each isolate were mapped against the Pt Race 1 reference genome; this high quality draft genome assembly includes a RNA-Seq based annotated gene set as well as the accompanying transcriptome and proteome studies based on the predicted products (Song et al., 2011;Bruce et al., 2014;Rampitsch et al., 2015;Cuomo et al., 2016). For the untranslated region (UTR), a detailed investigation of UTR length in Pt has not been carried out, so the selection of 1000 bases upstream and downstream of the coding sequences (CDS) was used to approximate UTRs, which may contain some intergenic sequences. ...
... For each isolate, between 74 and 81% of the sequence reads were mapped to the race 1 genome, which covered between 97.3 and 98.5% of the reference genome bases. Our subsequent analysis focused on the alignment data against this reference genome, as it not only covered the major part of our data, but this sequence also had annotations with transcriptome and proteome studies that facilitated further biological interpretations (Song et al., 2011;Bruce et al., 2014;Rampitsch et al., 2015). ...
... Of these 18 candidates, two could be functionally annotated by GO (PTTG_01476 involved in peptidase activity and PTTG_03866 involved in carbohydrate metabolic process) and eight were previously predicted as effectors by a transcriptome study on six races of Pt (Table 4; Bruce et al., 2014). Among these eight genes, PTTG_06324 was also supported by proteomic studies (Song et al., 2011;Rampitsch et al., 2015). As candidate effectors are also expected to be specific to individual species or strains, we examined the conservation of these genes in related fungi; 11 of the 18 candidates were conserved in other Basidiomycete genomes, 4 were Pt specific, and 3 were Puccinia specific (Table 4; Li et al., 2003;Cuomo et al., 2016). ...
Leaf rust is one of the most common and damaging diseases of wheat, and is caused by an obligate biotrophic basidiomycete, Puccinia triticina (Pt). In the present study, 20 Pt isolates from Australia, comprising 10 phenotype-matched pairs with contrasting pathogenicity for Lr20, were analyzed using whole genome sequencing. Compared to the reference genome of the American Pt isolate 1-1 BBBD Race 1, an average of 404,690 single nucleotide polymorphisms (SNPs) per isolate was found and the proportion of heterozygous SNPs was above 87% in the majority of the isolates, demonstrating a high level of polymorphism and a high rate of heterozygosity. From the genome-wide SNPs, a phylogenetic tree was inferred, which consisted of a large clade of 15 isolates representing diverse presumed clonal lineages including 14 closely related isolates and the more diverged isolate 670028, and a small clade of five isolates characterized by lower heterozygosity level. Principle component analysis detected three distinct clusters, corresponding exactly to the two major subsets of the small clade and the large clade comprising all 15 isolates without further separation of isolate 670028. While genome-wide association analysis identified 302 genes harboring at least one SNP associated with Lr20 virulence (p < 0.05), a Wilcoxon rank sum test revealed that 36 and 68 genes had significant (p < 0.05) and marginally significant (p < 0.1) differences in the counts of non-synonymous mutations between Lr20 avirulent and virulent groups, respectively. Twenty of these genes were predicted to have a signal peptide without a transmembrane segment, and hence identified as candidate effector genes corresponding to Lr20. SNP analysis also implicated the potential involvement of epigenetics and small RNA in Pt pathogenicity. Future studies are thus warranted to investigate the biological functions of the candidate effectors as well as the gene regulation mechanisms at epigenetic and post-transcription levels. Our study is the first to integrate phenotype-genotype association with effector prediction in Pt genomes, an approach that may circumvent some of the technical difficulties in working with obligate rust fungi and accelerate avirulence gene identification.
... An innovative technique used purified Puccinia triticina haustoria to generate a monoclonal antibody in mice that was subsequently used to isolate to near homogeneity by immunoprecipitation, haustoria from Puccinia triticina-infected wheat leaves. These antibody-purified Puccinia triticina haustoria revealed a content of 1,192 identified proteins, among which 140 candidate secreted effector proteins (CSEPs) (Rampitsch et al. 2015). When relative protein amounts among those 140 CSEPs is compared with the normalized transcript levels of the corresponding genes in wheat leaves 6 days after infection with Puccinia triticina, discrepancies between correlations of up to 2 orders of magnitude are observed for certain genes (G. ...
... This work finally proved the molecular basis of the gene-for-gene hypothesis as proposed by Flor in 1947(reviewed in Flor 1971) in the original model flax-flax rust pathosystem and was a first for rust fungi. Since haustoria are thought to be central hubs of the biotrophic interaction with crucial metabolite exchange and effector secretion, their isolation has helped to identify suites of effectors using ESTs and transcriptomic data (Catanzariti et al. 2006;Hahn and Mendgen 1997;Thara et al. 2003), and proteomics (Cooper et al. 2016;Rampitsch et al. 2015). Moreover, with the extensive genomic resources available, machine-learning algorithms tuned to rust fungal genes have been developed to generate lists of potential effectors, including their predicted localization (de Carvalho et al. 2017;Petre et al. 2014;Saunders et al. 2012;Sperschneider et al. 2017Sperschneider et al. , 2018a. ...
Among the thousands of rust species described, many are known for their devastating effects on their hosts which include major agriculture crops and trees. Hence, for over a century, these basidiomycete pathogenic fungi have been researched and experimented with. However, due to their biotrophic nature, they are challenging organisms to work with and, needing their hosts for propagation, represent pathosystems that are not easily experimentally accessible. Indeed, efforts to perform genetics have been few and far apart for the rust fungi, though one study performed in the 1940s was famously instrumental in formulating the gene-for-gene hypothesis describing pathogen-host interactions. By taking full advantage of the molecular genetic tools developed in the 1980s, research on many plant pathogenic microbes thrived, yet similar work on the rusts remained very challenging though not without some successes. However, the genomics era brought real breakthrough research for the biotrophic fungi and with innovative experimentation and the use of heterologous systems, molecular genetic analyses over the last two decades have significantly advanced our insight into the function of many rust fungus genes and their role in the interaction with their hosts. This has allowed optimizing efforts for resistance breeding and the design and testing of various novel strategies to reduce the devastating diseases they cause.
... A monoclonal antibody in mice was created using purified haustoria isolated from Pt-infected leaves. Purified haustoria contained 1,192 proteins, including 140 candidate secreted effector proteins (Rampitsch et al., 2015). In the haustorial transcriptome of Pgt, 520 secreted proteins were found, including 430 haustorially elevated secreted proteins and 90 genes expressed in germinated spores and haustoria as well as in haustoria alone (Cuomo et al., 2017). ...
Biotrophic plant pathogenic fungi are among the dreadful pathogens that continuously threaten the production of economically important crops. The interaction of biotrophic fungal pathogens with their hosts necessitates the development of unique infection mechanisms and involvement of various virulence-associated components. Biotrophic plant pathogenic fungi have an exceptional lifestyle that supports nutrient acquisition from cells of a living host and are fully dependent on the host for successful completion of their life cycle. The haustorium, a specialized infection structure, is the key organ for biotrophic fungal pathogens. The haustorium is not only essential in the uptake of nutrients without killing the host, but also in the secretion and delivery of effectors into the host cells to manipulate host immune system and defense responses and reprogram the metabolic flow of the host. Although there is a number of unanswered questions in this area yet, results from various studies indicate that the haustorium is the root of biotrophic fungal pathogens. This review provides an overview of current knowledge of the haustorium, its structure, composition, and functions, which includes the most recent haustorial transcriptome studies.
... They established that AvrLr2a canditae GN104ID162_007386, AvrLrka candidate GN1041D162_024924, and the AvrLr26 candidate GN104ID162_020918 had corresponding orthologs of PTTG_07365, PTTG_28070, and PTTG_1194 in the Pt race 1 genome, respectively (Wu et al., 2020). In support of their findings, a proteomics study of Pt race 1 haustoria predicted that these Pt race 1 orthologs were also candidate effectors (Rampitsch et al., 2015). ...
Biotrophic plant pathogenic fungi are widely distributed and are among the most damaging pathogenic organisms of agriculturally important crops responsible for significant losses in quality and yield. However, the pathogenesis of obligate parasitic pathogenic microorganisms is still under investigation because they cannot reproduce and complete their life cycle on an artificial medium. The successful lifestyle of biotrophic fungal pathogens depends on their ability to secrete effector proteins to manipulate or evade plant defense response. By integrating genomics, transcriptomics, and effectoromics, insights into how the adaptation of biotrophic plant fungal pathogens adapt to their host populations can be gained. Efficient tools to decipher the precise molecular mechanisms of rust-plant interactions, and standardized routines in genomics and functional pipelines have been established and will pave the way for comparative studies. Deciphering fungal pathogenesis not only allows us to better understand how fungal pathogens infect host plants but also provides valuable information for plant diseases control, including new strategies to prevent, delay, or inhibit fungal development. Our review provides a comprehensive overview of the efforts that have been made to decipher the effector proteins of biotrophic fungal pathogens and demonstrates how rapidly research in the field of obligate biotrophy has progressed.
... The development of a method to isolate haustoria from infected tissues was a remarkable finding in efforts to understand haustorial function and identify haustorial-specific genes [14,44]. More recently, another method used a direct proteomics approach to identify effector candidates from P. triticina Race 1 haustoria isolated with a specific monoclonal antibody [45]. Numerous effector proteins from rust fungi have been identified and characterized [46][47][48][49][50]. ...
Phakopsora pachyrhizi, which causes Asian soybean rust (ASR), secretes effector proteins to manipulate host immunity and promote disease. To date, only a small number of effectors have been identified from transcriptome studies. To obtain a more comprehensive understanding of P. pachyrhizi candidate secreted effector proteins (CSEPs), we sequenced the transcriptome using two next-generation sequencing technologies. Short-read Illumina RNA-Seq data was used for reducing base-calling errors for long-read PacBio Iso-Seq. After initial de novo assemblies for RNA-seq and error correction of transcripts for Iso-Seq followed by filtering, we obtained 8,528, 27,647, 26,895, and 17,141 non-plant, non-soybean transcripts at 3, 7, 10, and 14 days after inoculation, respectively. We identified a repertoire of CSEPs of which a majority was expressed during the later stages of infection, and many that could be bioinformatically associated with haustoria. This approach for identifying CSEPs improves our current understanding of the P. pachyrhizi effectorome, and these CSEPs are expected to be a valuable resource for future studies of P. pachyrhizi-soybean interactions.
... Elution from the column with 5% (v/v) β-mercaptoethanol yielded pools of enriched tryptic peptides whose Cys residues were oxidized prior to extraction and contained a mercaptoethanol adduct. These were analysed by LC-MS/MS using a quadrupole time-of-flight (QTOF) mass spectrometer (TripleTOF 5600: ABI Sciex, Concord, ON, Canada) under a fee-for-service contract at the Manitoba Centre for Proteomics and Systems Biology (Winnipeg, MB, Canada), as described previously (Rampitsch et al., 2015). Data analysis using Mascot was performed as described above, except that the variable modifications were set to: carbamidomethyl (Cys), destreak (Cys; with a mass shift of +76 atomic mass units) and oxidation (Met). ...
NADPH oxidase (NOX) is one of the sources of reactive oxygen species (ROS) that modulates the activity of proteins through modifications of their cysteine residues. Our previous study demonstrated the importance of NOX in both in the development and pathogenicity of the phytopathogen Fusarium graminearum. Comparative proteomics between wildtype and a Nox mutant of F. graminearum has now been used to identify active cysteine residues on candidate redox‐sensing proteins. A 2D‐gel approach based on labelling with monobromobimane (mBBr) identified 19 candidate proteins and was complemented with a gel‐free shotgun approach based on a biotin switch method, which yielded 99 candidates. The results indicated that in addition to temporal regulation, a large number of primary metabolic enzymes are potentially targeted by NoxAB‐generated ROS. Targeted disruption of these metabolic genes showed that while some are dispensable, others are essential. In addition to metabolic enzymes, developmental proteins such as the Woronin body major protein (FGSG_00837) and a GPI‐anchored protein (FGSG_10089) were also identified. Deleting either of these genes reduced the virulence of F. graminearum. Furthermore changing the redox modified cysteine (Cys³²⁵) residue in FGSG_10089 to either Serine or Phenylalanine results in similar phenotype to the FGSG_10089 knock‐out strain that displayed reduced virulence and altered cell wall morphology; this underscores the importance of Cys³²⁵ to the function of the protein. Our results indicated that NOX‐generated ROS act as intracellular signals in F. graminearum and that they modulate the activity of proteins affecting development and virulence in planta.
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... As moléculas ionizadas ganham ou perdem suas cargas por protonação, desprotonação ou ejeção de elétrons. São eletrostaticamente impulsionadas para dentro do aparelho e detectadas de acordo com sua massa por relação de carga (53). De cordo com KANG et al.; (39), quando plântulas de trigo foram tratadas com 0,5 mM de ácido salicílico (AS), 250 mM NaCl e 250 mM NaCl+0,5 mM AS para avaliar a influência do AS nos mecanismos de tolerância ao sal em trigo e um total de 39 proteínas diferencialmente reguladas para ambos sal e AS foram reveladas por 2D PAGE e 38 proteínas foram identificadas por MALDI-TOF/TOF MS. ...
RESUMO O agente causal da ferrugem da folha do trigo é o fungo Puccinia triticina. Essa doença causa danos elevados que podem comprometer a produtividade da cultura do trigo em até 80%, quando a infecção é intensa antes do florescimento e do enchimento de grãos. A utilização de cultivares resistentes é a melhor estratégia de controle da ferrugem da folha. Porém, devido à variabilidade do patógeno, a resistência dos genótipos é superada em até três anos após o seu lançamento. Para se defender da infecção do patógeno a planta desencadeia mecanismos de defesa, os quais têm a finalidade de evitar que o fungo colonize os tecidos do hospedeiro. Esses mecanismos de defesa podem estar associados com a expressão de genes que possuem a função de codificar proteínas envolvidas na resistência. Esta revisão discute a importância da interação planta-patógeno bem como das proteínas envolvidas. Também apresenta as principais técnicas de proteômica que visam identificar e quantificar as diferentes proteínas expressas nas células vegetais.
... This is generally assumed to be accomplished through the action of Nterminal signal or transit peptides, which are cleaved off to create the mature effector proteins. Effector protein candidates can be detected using a bioinformatics approach, however, only known sequences can be used since no universal signalling mechanism has been found in rusts (Rampitsch et al. 2015). In the current study, we analysed all fungal proteins bioinfomatically to detect effector candidates. ...
Over the last decade, comparative molecular
profiling studies between compatible and incompatible
plant-pathogen interactions have shown that susceptible
response of the host to a pathogen requires factors that
promote disease development. In this study, we examined
proteome profiles during a compatible interaction
between wheat and stripe rust. A 2D-LC system
(ProteomeLab PF2D) was used for protein separation
and to compare the proteome from infected and control
samples. More than 700 protein peaks at each time point
were compared between pathogen- and mockinoculated
samples. Selected proteins, with significant
differences in abundance were identified by nanoLCESI-
MS/MS and generated spectra were searched
against the wheat protein databases from UniProt, and
NCBI and the Puccinia database from The Broad Institute.
In total, the identified proteins comprised of 62 %
wheat and 38 % Pst proteins. All identified proteins
were searched by bioinformatics-based algorithms to
detect their subcellular localization and signal peptide
motifs which have the potential to catch the candidate
effector proteins. The wheat proteins were classified
based on their function. Although a compatible interaction,
many wheat proteins, such as antioxidants, PRs
and cold-responsive proteins, are implicated in defense
and stress tolerance. On the pathogen side, 64 proteins
were identified, and included some important pathogenicity
proteins that can play role in pathogen virulence
and suppress the host defense. In addition, we discovered
that nine proteins have a signal sequence and three
of the hypothetical fungal proteins, PGTG_11681T0,
PGTG_07231T0 and CBH50687.1, have been tentatively
identified as candidate effectors.
... This is generally assumed to be accomplished through the action of Nterminal signal or transit peptides, which are cleaved off to create the mature effector proteins. Effector protein candidates can be detected using a bioinformatics approach, however, only known sequences can be used since no universal signalling mechanism has been found in rusts (Rampitsch et al. 2015). In the current study, we analysed all fungal proteins bioinfomatically to detect effector candidates. ...
Over the last decade, comparative molecular profiling studies between compatible and incompatible plant-pathogen interactions have shown that susceptible response of the host to a pathogen requires factors that promote disease development. In this study, we examined proteome profiles during a compatible interaction between wheat and stripe rust. A 2D-LC system (ProteomeLab PF2D) was used for protein separation and to compare the proteome from infected and control samples. More than 700 protein peaks at each time point were compared between pathogen- and mock-inoculated samples. Selected proteins, with significant differences in abundance were identified by nanoLC-ESI- MS/MS and generated spectra were searched against the wheat protein databases from UniProt, and NCBI and the Puccinia database from The Broad Institute. In total, the identified proteins comprised of 62 % wheat and 38 % Pst proteins. All identified proteins were searched by bioinformatics-based algorithms to detect their subcellular localization and signal peptide motifs which have the potential to catch the candidate effector proteins. The wheat proteins were classified based on their function. Although a compatible interaction, many wheat proteins, such as antioxidants, PRs and cold-responsive proteins, are implicated in defense and stress tolerance. On the pathogen side, 64 proteins were identified, and included some important pathogenicity proteins that can play role in pathogen virulence and suppress the host defense. In addition, we discovered that nine proteins have a signal sequence and three of the hypothetical fungal proteins, PGTG_11681T0, PGTG_07231T0 and CBH50687.1, have been tentatively identified as candidate effectors.
... It was clear that more work would be needed to provide details on the biochemistry of this pathosystem. This work led to the production of monoclonal antibodies to enable purification of Race 1 (BBBD) haustoria to near-homogeneity (Rampitsch et al. 2015). The isolation of the haustorial proteome of this race can now be used in conjunction with the sequenced genome to identify proteins and genes which could be involved in pathogenesis. ...
Wheat leaf rust, caused by Puccinia triticina Eriks., is of worldwide concern for wheat producers. The disease has been an annual problem for Canadian wheat producers since the early days of wheat cultivation in the 1800s, and research focused on combating this disease began in the early 1900s. Significant progress was made towards understanding the epidemiology of wheat leaf rust and developing genetic resistance in many countries worldwide. This review paper focuses exclusively on the research and development done in whole, or in part, in Canada. An integrated approach to controlling wheat leaf rust consisted of research in the following areas: the early research on wheat leaf rust in Canada, breeding and commercialization of high quality rust resistant wheat cultivars, discovery and genetic analysis of leaf rust resistance genes, the population biology and genetics of the P. triticina/wheat interaction. This review summarizes the research in each of these areas and the connections between the different aspects of the research. A multi-disciplinary team approach has been key to the advancements made within these diverse research fields in Canada since the early 1900s.
... This is generally assumed to be accomplished through the action of Nterminal signal or transit peptides, which are cleaved off to create the mature effector proteins. Effector protein candidates can be detected using a bioinformatics approach, however, only known sequences can be used since no universal signalling mechanism has been found in rusts (Rampitsch et al. 2015). In the current study, we analysed all fungal proteins bioinfomatically to detect effector candidates. ...
Powdery mildew, a fungal pathogen, poses significant threats to various plant species, including economically important ones. Conventional treatment methods involving compound fungicides, while common, raise environmental and social health concerns due to their potential adverse effects. Recent years have witnessed the rise of omics technologies and the innovative CRISPR-Cas9 gene editing system, drawing considerable attention as potential tools for disease management. This review aims to provide a comprehensive understanding of how
omics and CRISPR-Cas9 are employed to unravel the molecular mechanisms of powdery mildew and to develop effective disease control strategies. The discussion delves into the utilization of genomics and transcriptomics methodologies to elucidate the intricate interactions between the powdery mildew pathogen and its host plants. Additionally, it explores the potential of CRISPR-Cas9 technology for targeted gene editing to enhance plant
resistance against powdery mildew. The review goes further to propose a practical strategy for implementing these methods in the management of powdery mildew, offering insights into the selection of target genes and refining CRISPR-Cas9 delivery mechanisms. Overall, the review underscores the significant promise of omics and CRISPR-Cas9 technologies in advancing our comprehension of powdery mildew pathogenesis and provides
valuable guidance on their application for efficient disease management.
Proteomik yaklaşımları 2000 li yılların başlarına kadar mikroorganizmalar ve hayvansal kaynaklı örneklerde ağırlıklı olarak kullanıldı. Bu dönemde bitki proteomik çalışmaları yok denecek kadar azdır. Bitkisel dokulardaki sert hücre çeperleri, karmaşık ve çok çeşitli sekonder metabolitlerin varlığı, fazla miktardaki pigmentler, proteazlar, polifenoller, polisakkaritler, nişasta ve lipitler total protein örneklerinin hazırlanması ve proteinlerin ayrımı sırasında pek çok soruna neden olmuştur. Ancak her bir sorunun üstesinden gelmek üzere sürdürülen çabalar sayesinde bitki dünyasında da proteomik yaklaşım kullanımı yaygınlaşmıştır. Bu derlemede, örnek hazırlığından protein tanımlamaya kadar tüm basamaklar yöntemsel gelişmeleri de kapsayacak şekilde ayrıntılı olarak ele alınmış ve konuyla ilgili araştırıcıların maksimum yararlanabileceği bir kaynak oluşturulmaya çalışılmıştır.
There are over 500 candidate secreted effector proteins (CSEPs) or Blumeria effector candidates (BECs) specific to the barley powdery mildew pathogen Blumeria graminis f.sp. hordei. The CSEP/ BEC proteins are expressed and predicted to be secreted by biotrophic feeding structures called haustoria. Eight BECs are required for the formation of functional haustoria. These include the RNAse-like effector BEC1054 (synonym CSEP0064). In order to identify host proteins targeted by BEC1054, recombinant BEC1054 was expressed in E. coli, solubilised and used in pull-down assays from total barley protein extracts. Many putative interactors were identified by LC-MS/MS, despite subtraction of unspecific binders in negative controls. Therefore, a directed yeast-2-hybrid assay, developed to measure the effectiveness of the interactions in yeast, was used to validate putative interactors. We conclude that BEC1054 may target several host proteins including a glutathione-S-transferase, a malate dehydrogenase and a pathogen-related-5 protein isoform, indicating a possible role for BEC1054 in compromising well known key players of resistance and response to pathogens. In addition, pull-down data indicated the possible interaction of BEC1054 with ribosomal proteins, including an elongation factor 1 gamma. This data supports the idea that BEC1054 binds to barley ribosomal proteins. The biological significance of this remains to be defined.
Leaf rust caused by Puccinia triticina is the most common and widely distributed of the three wheat rusts. Losses from leaf rust are usually less damaging than
those from stem rust and stripe rust, but leaf rust causes greater annual losses due to its more frequent and widespread occurrence.
Yield losses from leaf rust are mostly due to reductions in kernel weight. Many laboratories worldwide conduct leaf rust surveys
and virulence analyses. Most currently important races (pathotypes) have either evolved through mutations in existing populations
or migrated from other, often unknown, areas. Several leaf rust resistance genes are cataloged, and high levels of slow rusting
adult plant resistance are available in high yielding CIMMYT wheats. This paper summarizes the importance of leaf rust in
the main wheat production areas as reflected by yield losses, the complexity of virulence variation in pathogen populations,
the role cultivars with race-specific resistance play in pathogen evolution, and the control measures currently practiced
in various regions of the world.
Keywords
Triticum aestivum
–
Triticum turgidum
–Resistance–Races
Fungal and oomycete plant parasites are among the most devastating pathogens of food crops. These microbes secrete effector proteins inside plant cells to manipulate host processes and facilitate colonization. How these effectors reach the host cytoplasm remains an unclear and debated area of plant research. In this article, we examine recent conflicting findings that have generated discussion in the field. We also highlight promising approaches based on studies of both parasite and host during infection. Ultimately, this knowledge may inform future broad spectrum strategies for protecting crops from such pathogens.
Haustoria of biotrophic rust fungi are responsible for the uptake of nutrients from their hosts and for the production of secreted proteins, known as effectors, which modulate the host immune system. The identification of the transcriptome of haustoria and an understanding of the functions of expressed genes therefore hold essential keys for the elucidation of fungus-plant interactions and the development of novel fungal control strategies. Here, we purified haustoria from infected leaves and used 454 sequencing to examine the haustorial transcriptomes of Phakopsora pachyrhizi and Uromyces appendiculatus, the causal agents of soybean rust and common bean rust, respectively. These pathogens cause extensive yield losses in their respective legume crop hosts. A series of analyses were used to annotate expressed sequences, including transposable elements and viruses, to predict secreted proteins from the assembled sequences and to identify families of candidate effectors. This work provides a foundation for the comparative analysis of haustorial gene expression with further insights into physiology and effector evolution.
Stripe rust caused by the fungus Puccinia striiformis f.sp. tritici (Pst) is a major constraint to wheat production worldwide. The molecular events that underlie Pst pathogenicity are largely unknown. Like all rusts, Pst creates a specialized cellular structure within host cells called the haustorium to obtain nutrients from wheat, and to secrete pathogenicity factors called effector proteins. We purified Pst haustoria and used next-generation sequencing platforms to assemble the haustorial transcriptome as well as the transcriptome of germinated spores. 12,282 transcripts were assembled from 454-pyrosequencing data and used as reference for digital gene expression analysis to compare the germinated uredinospores and haustoria transcriptomes based on Illumina RNAseq data. More than 400 genes encoding secreted proteins which constitute candidate effectors were identified from the haustorial transcriptome, with two thirds of these up-regulated in this tissue compared to germinated spores. RT-PCR analysis confirmed the expression patterns of 94 effector candidates. The analysis also revealed that spores rely mainly on stored energy reserves for growth and development, while haustoria take up host nutrients for massive energy production for biosynthetic pathways and the ultimate production of spores. Together, these studies substantially increase our knowledge of potential Pst effectors and provide new insights into the pathogenic strategies of this important organism.
Background
Wheat yellow (stripe) rust caused by Puccinia striiformis f. sp. tritici (PST) is one of the most devastating diseases of wheat worldwide. To design effective breeding strategies that maximize the potential for durable disease resistance it is important to understand the molecular basis of PST pathogenicity. In particular, the characterisation of the structure, function and evolutionary dynamics of secreted effector proteins that are detected by host immune receptors can help guide and prioritize breeding efforts. However, to date, our knowledge of the effector repertoire of cereal rust pathogens is limited.
Results
We re-sequenced genomes of four PST isolates from the US and UK to identify effector candidates and relate them to their distinct virulence profiles. First, we assessed SNP frequencies between all isolates, with heterokaryotic SNPs being over tenfold more frequent (5.29 ± 2.23 SNPs/kb) than homokaryotic SNPs (0.41 ± 0.28 SNPs/kb). Next, we implemented a bioinformatics pipeline to integrate genomics, transcriptomics, and effector-focused annotations to identify and classify effector candidates in PST. RNAseq analysis highlighted transcripts encoding secreted proteins that were significantly enriched in haustoria compared to infected tissue. The expression of 22 candidate effector genes was characterised using qRT-PCR, revealing distinct temporal expression patterns during infection in wheat. Lastly, we identified proteins that displayed non-synonymous substitutions specifically between the two UK isolates PST-87/7 and PST-08/21, which differ in virulence to two wheat varieties. By focusing on polymorphic variants enriched in haustoria, we identified five polymorphic effector candidates between PST-87/7 and PST-08/21 among 2,999 secreted proteins. These allelic variants are now a priority for functional validation as virulence/avirulence effectors in the corresponding wheat varieties.
Conclusions
Integration of genomics, transcriptomics, and effector-directed annotation of PST isolates has enabled us to move beyond the single isolate-directed catalogues of effector proteins and develop a framework for mining effector proteins in closely related isolates and relate these back to their defined virulence profiles. This should ultimately lead to more comprehensive understanding of the PST pathogenesis system, an important first step towards developing more effective surveillance and management strategies for one of the most devastating pathogens of wheat.
About 8,000 years ago in the Fertile Crescent, a spontaneous hybridization of the wild diploid grass Aegilops tauschii (2n = 14; DD) with the cultivated tetraploid wheat Triticum turgidum (2n = 4x = 28; AABB) resulted in hexaploid wheat (T. aestivum; 2n = 6x = 42; AABBDD). Wheat has since become a primary staple crop worldwide as a result of its enhanced adaptability to a wide range of climates and improved grain quality for the production of baker's flour. Here we describe sequencing the Ae. tauschii genome and obtaining a roughly 90-fold depth of short reads from libraries with various insert sizes, to gain a better understanding of this genetically complex plant. The assembled scaffolds represented 83.4% of the genome, of which 65.9% comprised transposable elements. We generated comprehensive RNA-Seq data and used it to identify 43,150 protein-coding genes, of which 30,697 (71.1%) were uniquely anchored to chromosomes with an integrated high-density genetic map. Whole-genome analysis revealed gene family expansion in Ae. tauschii of agronomically relevant gene families that were associated with disease resistance, abiotic stress tolerance and grain quality. This draft genome sequence provides insight into the environmental adaptation of bread wheat and can aid in defining the large and complicated genomes of wheat species.
Rust fungi are obligate biotrophic pathogens that cause considerable damage on crop plants. Puccinia graminis f. sp. tritici, the causal agent of wheat stem rust, and Melampsora larici-populina, the poplar leaf rust pathogen, have strong deleterious impacts on wheat and poplar wood production, respectively. Filamentous pathogens such as rust fungi secrete molecules called disease effectors that act as modulators of host cell physiology and can suppress or trigger host immunity. Current knowledge on effectors from other filamentous plant pathogens can be exploited for the characterisation of effectors in the genome of recently sequenced rust fungi. We designed a comprehensive in silico analysis pipeline to identify the putative effector repertoire from the genome of two plant pathogenic rust fungi. The pipeline is based on the observation that known effector proteins from filamentous pathogens have at least one of the following properties: (i) contain a secretion signal, (ii) are encoded by in planta induced genes, (iii) have similarity to haustorial proteins, (iv) are small and cysteine rich, (v) contain a known effector motif or a nuclear localization signal, (vi) are encoded by genes with long intergenic regions, (vii) contain internal repeats, and (viii) do not contain PFAM domains, except those associated with pathogenicity. We used Markov clustering and hierarchical clustering to classify protein families of rust pathogens and rank them according to their likelihood of being effectors. Using this approach, we identified eight families of candidate effectors that we consider of high value for functional characterization. This study revealed a diverse set of candidate effectors, including families of haustorial expressed secreted proteins and small cysteine-rich proteins. This comprehensive classification of candidate effectors from these devastating rust pathogens is an initial step towards probing plant germplasm for novel resistance components.
Puccinia triticina (Pt) is a representative of several cereal-infecting rust fungal pathogens of major economic importance world wide. Upon entry through leaf stomata, these fungi establish intracellular haustoria, crucial feeding structures. We report the first proteome of infection structures from parasitized wheat leaves, enriched for haustoria through filtration and sucrose density centrifugation. 2-D PAGE MS/MS and gel-based LC-MS (GeLC-MS) were used to separate proteins. Generated spectra were compared with a partial proteome predicted from a preliminary Pt genome and generated ESTs, to a comprehensive genome-predicted protein complement from the related wheat stem rust fungus, Puccinia graminis f. sp. tritici (Pgt) and to various plant resources. We identified over 260 fungal proteins, 16 of which matched peptides from Pgt. Based on bioinformatic analyses and/or the presence of a signal peptide, at least 50 proteins were predicted to be secreted. Among those, six have effector protein signatures, some are related and the respective genes of several seem to belong to clusters. Many ribosomal structural proteins, proteins involved in energy, general metabolism and transport were detected. Measuring gene expression over several life cycle stages of ten representative candidates using quantitative RT-PCR, all were shown to be strongly upregulated and four expressed solely upon infection.
Hydrogen peroxide (H2O2) is recognized as an important signalling molecule. There are two important aspects to this function: H2O2 production and its diffusion to its sites of action. The production of H2O2 by photosynthetic electron transport and its ability to diffuse through the chloroplast envelope membranes has been investigated
using spin trapping electron paramagnetic resonance spectroscopy and H2O2-sensitive fluorescence dyes. It was found that, even at low light intensity, a portion of H2O2 produced inside the chloroplasts can leave the chloroplasts thus escaping the effective antioxidant systems located inside
the chloroplast. The production of H2O2 by chloroplasts and the appearance of H2O2 outside chloroplasts increased with increasing light intensity and time of illumination. The amount of H2O2 that can be detected outside the chloroplasts has been shown to be up to 5% of the total H2O2 produced inside the chloroplasts at high light intensities. The fact that H2O2 produced by chloroplasts can be detected outside these organelles is an important finding in terms of understanding how chloroplastic
H2O2 can serve as a signal molecule.
To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity.
A number of fungal and oomycete plant pathogens of major economic importance feed on their hosts by means of haustoria, which they place inside living plant cells. The underlying mechanisms are poorly understood, partly due to difficulty in preparing haustoria. We have therefore developed a procedure for isolating haustoria from the barley powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh). We subsequently aimed to understand the molecular mechanisms of haustoria through a study of their proteome. Extracted proteins were digested using trypsin, separated by LC, and analysed by MS/MS. Searches of a custom Bgh EST sequence database and the NCBI-NR fungal protein database, using the MS/MS data, identified 204 haustoria proteins. The majority of the proteins appear to have roles in protein metabolic pathways and biological energy production. Surprisingly, pyruvate decarboxylase (PDC), involved in alcoholic fermentation and commonly abundant in fungi and plants, was absent in our Bgh proteome data set. A sequence encoding this enzyme was also absent in our EST sequence database. Significantly, BLAST searches of the recently available Bgh genome sequence data also failed to identify a sequence encoding this enzyme, strongly indicating that Bgh does not have a gene for PDC.
It is accepted that most fungal avirulence genes encode virulence factors that are called effectors. Most fungal effectors are secreted, cysteine-rich proteins, and a role in virulence has been shown for a few of them, including Avr2 and Avr4 of Cladosporium fulvum, which inhibit plant cysteine proteases and protect chitin in fungal cell walls against plant chitinases, respectively. In resistant plants, effectors are directly or indirectly recognized by cognate resistance proteins that reside either inside the plant cell or on plasma membranes. Several secreted effectors function inside the host cell, but the uptake mechanism is not yet known. Variation observed among fungal effectors shows two types of selection that appear to relate to whether they interact directly or indirectly with their cognate resistance proteins. Direct interactions seem to favor point mutations in effector genes, leading to amino acid substitutions, whereas indirect interactions seem to favor jettison of effector genes.
Plant-associated organisms secrete proteins and other molecules to modulate plant defense circuitry and enable colonization of plant tissue. Understanding the molecular function of these secreted molecules, collectively known as effectors, became widely accepted as essential for a mechanistic understanding of the processes underlying plant colonization. This review summarizes recent findings in the field of effector biology and highlights the common concepts that have emerged from the study of cellular plant pathogen effectors.
Levels of the C6-polyol mannitol were observed to rise dramatically in the biotrophic interaction of the rust fungus Uromyces fabae and its host plant Vicia faba. Mannitol was found in millimolar concentrations in extracts and apoplastic fluids of infected leaves and also in extracts of spores. We suggest that this polyol might have at least a dual function: first, as a carbohydrate storage compound, and second, as a scavenger of reactive oxygen species. Mannitol accumulation is accompanied by high expression of a mannitol dehydrogenase (MAD1) in haustoria. While MAD1 transcripts were detected in haustoria only, immunolocalization studies show that the gene product is also present in spores. Kinetic and thermodynamic analyses of the MAD1p catalyzed reactions indicate that the enzyme might be responsible for the production of mannitol in haustoria and for the utilization of mannitol in spores. Since V. faba is normally unable to synthesize or utilize polyols, the multipurpose usage of mannitol seems an ideal strategy for the fungal pathogen.
We present here Blast2GO (B2G), a research tool designed with the main purpose of enabling Gene Ontology (GO) based data mining on sequence data for which no GO annotation is yet available. B2G joints in one application GO annotation based on similarity searches with statistical analysis and highlighted visualization on directed acyclic graphs. This tool offers a suitable platform for functional genomics research in non-model species. B2G is an intuitive and interactive desktop application that allows monitoring and comprehension of the whole annotation and analysis process.
Availability:
Blast2GO is freely available via Java Web Start at http://www.blast2go.de.
Supplementary material:
http://www.blast2go.de -> Evaluation.
The production of reactive oxygen species (ROS), via consumption of oxygen in a so-called oxidative burst, is one of the earliest cellular responses following successful pathogen recognition. Apoplastic generation of superoxide (O2−), or its dismutation product hydrogen peroxide (H2O2), has been documented following recognition of a variety of pathogens (Doke, 1983; Auh and Murphy, 1995; Grant et al., 2000b). Avirulent pathogens, successfully recognized via the action of disease resistance (R) gene products in plant immune system, elicit a biphasic ROS accumulation with a low-amplitude, transient first phase, followed by a sustained phase of much higher magnitude that correlates with disease resistance (Lamb and Dixon, 1997). However, virulent pathogens that avoid host recognition induce only the transient, low-amplitude first phase of this response, suggesting a role for ROS in the establishment of the defenses. In line with this conclusion, elicitors of defense responses, often referred to as microbe-associated molecular patterns (MAMPs), also trigger an oxidative burst. Initial characterization of the oxidative burst left unclear whether ROS acted as executioners of pathogen, host cells (in the form of the familiar hypersensitive response [HR]), or both, or, alternatively, as signaling molecules that were not directly involved in the mechanisms that actually stopped pathogen growth.
In the plant cell, ROS can directly cause strengthening of host cell walls via cross-linking of glycoproteins (Bradley et al., 1992; Lamb and Dixon, 1997), or lipid peroxidation and membrane damage (Lamb and Dixon, 1997; Montillet et al., 2005). However, it is also evident that ROS are important signals mediating defense gene activation (Levine et al., 1994). Additional regulatory functions for ROS in defense occur in conjunction with other plant signaling molecules, particularly with salicylic acid (SA) and nitric oxide (NO; see Fig. 1). However, ROS also regulate additional plant responses in relation to other signals. Here, we discuss these roles of ROS with a focus on the response to pathogen infection.
Figure 1.
ROS production and functions in response to pathogens.
Powdery mildews, obligate biotrophic fungal parasites on a wide range of important crops, can be controlled by plant resistance (R) genes, but these are rapidly overcome by parasite mutants evading recognition. It is unknown how this rapid evolution occurs without apparent loss of parasite fitness. R proteins recognize avirulence (AVR) molecules from parasites in a gene-for-gene manner and trigger defense responses. We identify AVR(a10) and AVR(k1) of barley powdery mildew fungus, Blumeria graminis f sp hordei (Bgh), and show that they induce both cell death and inaccessibility when transiently expressed in Mla10 and Mlk1 barley (Hordeum vulgare) varieties, respectively. In contrast with other reported fungal AVR genes, AVR(a10) and AVR(k1) encode proteins that lack secretion signal peptides and enhance infection success on susceptible host plant cells. AVR(a10) and AVR(k1) belong to a large family with >30 paralogues in the genome of Bgh, and homologous sequences are present in other formae speciales of the fungus infecting other grasses. Our findings imply that the mildew fungus has a repertoire of AVR genes, which may function as effectors and contribute to parasite virulence. Multiple copies of related but distinct AVR effector paralogues might enable populations of Bgh to rapidly overcome host R genes while maintaining virulence.
Many plant-associated microbes are pathogens that impair plant growth and reproduction. Plants respond to infection using a two-branched innate immune system. The first branch recognizes and responds to molecules common to many classes of microbes, including non-pathogens. The second responds to pathogen virulence factors, either directly or through their effects on host targets. These plant immune systems, and the pathogen molecules to which they respond, provide extraordinary insights into molecular recognition, cell biology and evolution across biological kingdoms. A detailed understanding of plant immune function will underpin crop improvement for food, fibre and biofuels production.
A major insight that has emerged in the study of haustoria-forming plant pathogens over the last few years is that these eukaryotic biotrophs deliver suites of secreted proteins into host cells during infection. This insight has largely derived from successful efforts to identify avirulence (Avr) genes and their products from these pathogens. These Avr genes, identified from a rust and a powdery mildew fungus and three oomycete species, encode small proteins that are recognized by resistance proteins in the host plant cytoplasm, suggesting that they are transported inside plant cells during infection. These Avr proteins probably represent examples of fungal and oomycete effector proteins with important roles in subverting host cell biology during infection. In this respect, they represent a new opportunity to understand the basis of disease caused by these biotrophic pathogens. Elucidating how these pathogen proteins gain entry into plant cells and their biological function will be key questions for future research.
Five unrelated avirulence (Avr) gene families have been cloned from flax rust and barley powdery mildew, fungal pathogens that make close contact with living host plant cells using specialized feeding structures called haustoria. Transgenic expression studies indicate Avr proteins are recognized by disease resistance (R) proteins within host cells, which suggests that Avr proteins are transported via an as yet unidentified route from the fungus to the host during infection. Recognition of flax rust AvrL567 proteins is by direct R-Avr protein interaction. Virulence effector functions have been demonstrated for barley powdery mildew Avr proteins Avra10 and Avrk1. Mildew resistance triggered by Avra10 in barley involves association of the cognate barley R protein Mla10 and transcriptional repressor proteins, including HvWRKY2, in the host nucleus. High amplitude defence gene expression has a dual dependence on transcriptional de-repression induced by specific R-Avr protein recognition and additionally, activation signals initiated by host perception of general pathogen molecules.
Reactive oxygen species (ROS) play an integral role as signalling molecules in the regulation of numerous biological processes
such as growth, development, and responses to biotic and/or abiotic stimuli in plants. To some extent, various functions of
ROS signalling are attributed to differences in the regulatory mechanisms of respiratory burst oxidase homologues (RBOHs)
that are involved in a multitude of different signal transduction pathways activated in assorted tissue and cell types under
fluctuating environmental conditions. Recent findings revealed that stress responses in plants are mediated by a temporal–spatial
coordination between ROS and other signals that rely on production of stress-specific chemicals, compounds, and hormones.
In this review we will provide an update of recent findings related to the integration of ROS signals with an array of signalling
pathways aimed at regulating different responses in plants. In particular, we will address signals that confer systemic acquired
resistance (SAR) or systemic acquired acclimation (SAA) in plants.
Various cytochemical tests on the wheat stem rust fungus were used to determine differences in components of the walls of the haustorium mother cell at the host cell penetration site and the haustorial neck and body and to describe some of the chemical properties of the extrahaustorial matrix. There were two transition zones with respect to wall composition. The first was at the host cell penetration site; chitin, present in haustorium mother cell walls, was not detected in haustorial neck walls. The second transition zone was at the neck ring; compared with walls of the proximal neck region, those distal to the neck ring contained more protein and lost much of their periodate – thiocarbohydrazide – silver proteinate reactive material and all concanavalin A binding material after treatment with protease. The two wall layers of the distal part of the haustorial neck were continuous with those of the haustorium; the wall layers of young haustorial bodies shared their staining properties and lectin affinities with those of the distal part of the haustorial necks, reflecting their common origin. As the haustoria matured, their body walls bound wheat germ lectin, but the neck walls did not. Tests indicated that polysaccharide and glycoprotein were present in the extrahaustorial matrix.
The morphology of infection structure development of Puccinia recondita f. sp. tritici on and in susceptible and resistant wheat lines inoculated with urediospores was examined by SEM. The germ-tube extends over the leaf surface and elongates perpendicularly to the long axis of the leaf. When the germ-tube encounters the stomatal lip, an appressorium forms over the stoma and the pore is entered by an infection peg produced on the surface of the appressorium in contact with the host leaf. At 6 h post-inoculation (hpi), infection pegs develop terminally substomatal vesicles (SSVs) in the substomatal chambers of all wheat lines. A septum separates each SSV from its interconnective tube. A primary infection hypha forms terminally from the elongated SSV either parallel to the long axis of the stomatal slit or perpendicular to the leaf surface. When a primary infection hypha attaches to a host cell, a septum forms cutting off the tip of the hypha, delimiting a terminal haustorium mother cell (HMC) by 12 hpi. Secondary infection hyphae arise from a position proximal to, and in the proximity of, the HMC septum. Additional HMCs are formed when a secondary hypha or a tertiary hypha adheres to a plant cell. Infection sites with HMCs were observed at 24 hpi and at subsequent sampling stages. There were no significant differences between the infection processes on the three wheat lines examined in this study.
Powdery mildews are phytopathogenic ascomycetes that have an obligate biotrophic lifestyle and establish intimate relationships with their plant hosts. A crucial aspect of this plant-fungus interaction is the formation of specialized fungal infection structures termed haustoria. Although located within the cell boundaries of plant epidermal cells, haustoria remain separated from the plant cytoplasm by a host plasma membrane derivative, the extrahaustorial membrane. Haustoria are thought to represent pivotal sites of nutrient uptake and effector protein delivery. We enriched haustorial complexes from Arabidopsis thaliana plants infected with the powdery mildew fungus Golovinomyces orontii and performed in-depth transcriptome analysis by 454-based pyrosequencing of haustorial cDNAs. We assembled 7077 expressed sequence tag (EST) contigs with greater than 5-fold average coverage and analyzed these with regard to the respective predicted protein functions. We found that transcripts coding for gene products with roles in protein turnover, detoxification of reactive oxygen species and fungal pathogenesis are abundant in the haustorial EST contigs, while surprisingly transcripts encoding presumptive nutrient transporters were not highly represented in the haustorial cDNA library. A substantial proportion (∼38%) of transcripts coding for predicted secreted proteins comprises effector candidates. Our data provide valuable insights into the transcriptome of the key infection structure of a model obligate biotrophic phytopathogen.
Haustoria of the wheat stem rust fungus,Puccinia graminis f.sp.tritici, race 32, were isolated from two different infected wheat cultivars. Yield of haustoria from the highly susceptible wheat cv. “Little Club” was 10 times higher than that of the resistant wheat line “Prelude × Eagle” carrying the Sr26 gene for resistance toP. graminis f.sp.tritici. Tests for the integrity of haustoria using the stain methylene blue showed that up to 91% of the haustoria were undamaged.
The study of plant disease and immunity is benefiting tremendously from proteomics. Parallel streams of research from model systems, from pathogens in vitro and from the relevant pathogen-crop interactions themselves have begun to reveal a model of how plants succumb to invading pathogens and how they defend themselves without the benefit of a circulating immune system. In this review, we discuss the contribution of proteomics to these advances, drawing mainly on examples from crop-fungus interactions, from Arabidopsis-bacteria interactions, from elicitor-based model systems and from pathogen studies, to highlight also the important contribution of non-crop systems to advancing crop protection.
Rust fungi form complex associations with infected plant cells through haustoria, specialized structures which function both in nutrient uptake and in the delivery of protein effectors into host cell. While first characterized as avirulence factors that are recognized by host immune receptors, rust effectors are believed to play important roles in establishing successful infection. Several rust avirulence and effector proteins have been identified as small secreted proteins that are expressed in haustoria, and these characteristics have been used in the systematic prediction of effector gene candidates from recently sequenced rust genomes. The poplar rust and wheat stem rust genomes for instance contain over a thousand small secreted proteins that may have effector functions. These proteins are highly divergent between these rust species, reflecting the rapid evolution expected of virulence factors that directly engage with the host organism. New approaches are being developed to facilitate the functional description of rust effectors and to define their contributions to rust infection.
Quantitative shotgun proteomics is dependent on the detection, identification, and quantitative analysis of peptides. An issue arises with peptides that are shared between multiple proteins. What protein did they originate from and how should these shared peptides be used in a quantitative proteomics workflow? To systematically evaluate shared peptides in label-free quantitative proteomics, we devised a well-defined protein sample consisting of known concentrations of six albumins from different species, which we added to a highly complex yeast lysate. We used the spectral counts based normalized spectral abundance factor (NSAF) as the starting point for our analysis and compared an exhaustive list of possible combinations of parameters to determine what was the optimal approach for dealing with shared peptides and shared spectral counts. We showed that distributing shared spectral counts based on the number of unique spectral counts led to the most accurate and reproducible results.
Haustoria are morphological features of an extremely successful class of plant parasites, the obligate biotrophs. The broad phylogenetic spectrum of organisms producing haustoria suggests that these structures have arisen many times in the course of evolution and represent specific adaptations of these organisms to the close interaction with their respective host plants. This close interaction and the fact that these structures cannot be produced in vitro have hampered an analysis of the roles of haustoria in biotrophy for many decades. Only recently has it become possible to analyse haustorial function at a molecular level. A picture is beginning to emerge indicating that haustoria do not only serve in nutrient uptake a task postulated for these elements ever since their discovery. Moreover, they seem to perform enormous biosynthetic duties. They also seem to be engaged in the suppression of host defense responses and in redirecting or reprogramming the hosts metabolic flow. This review intends to summarize current knowledge about the structure and function especially of rust haustoria.
Unlabelled:
Leaf rust, caused by Puccinia triticina, is the most common rust disease of wheat. The fungus is an obligate parasite capable of producing infectious urediniospores as long as infected leaf tissue remains alive. Urediniospores can be wind-disseminated and infect host plants hundreds of kilometres from their source plant, which can result in wheat leaf rust epidemics on a continental scale. This review summarizes current knowledge of the P. triticina/wheat interaction with emphasis on the infection process, molecular aspects of pathogenicity, rust resistance genes in wheat, genetics of the host parasite interaction, and the population biology of P. triticina.
Taxonomy:
Puccinia triticina Eriks.: kingdom Fungi, phylum Basidiomycota, class Urediniomycetes, order Uredinales, family Pucciniaceae, genus Puccinia.
Host range:
Telial/uredinial (primary) hosts: common wheat (Triticum aestivum L.), durum wheat (T. turgidum L. var. durum), cultivated emmer wheat (T. dicoccon) and wild emmer wheat (T. dicoccoides), Aegilops speltoides, goatgrass (Ae. cylindrica), and triticale (X Triticosecale). Pycnial/aecial (alternative) hosts: Thalictrum speciosissimum (= T. flavum glaucum) and Isopyrum fumaroides.
Identification:
Leaf rust is characterized by the uredinial stage. Uredinia are up to 1.5 mm in diameter, erumpent, round to ovoid, with orange to brown uredinia that are scattered on both the upper and the lower leaf surfaces of the primary host. Uredinia produce urediniospores that are sub-globoid, average 20 microm in diameter and are orange-brown, with up to eight germ pores scattered in thick, echinulate walls.
Disease symptoms:
Wheat varieties that are fully susceptible have large uredinia without causing chlorosis or necrosis in the host tissues. Resistant wheat varieties are characterized by various responses from small hypersensitive flecks to small to moderate size uredinia that may be surrounded by chlorotic and/or necrotic zones.
Useful website:
USDA Cereal Disease Laboratory: http://www.ars.usda.gov/mwa/cdl.
A neural network-based tool, TargetP, for large-scale subcellular location prediction of newly identified proteins has been developed. Using N-terminal sequence information only, it discriminates between proteins destined for the mitochondrion, the chloroplast, the secretory pathway, and "other" localizations with a success rate of 85% (plant) or 90% (non-plant) on redundancy-reduced test sets. From a TargetP analysis of the recently sequenced Arabidopsis thaliana chromosomes 2 and 4 and the Ensembl Homo sapiens protein set, we estimate that 10% of all plant proteins are mitochondrial and 14% chloroplastic, and that the abundance of secretory proteins, in both Arabidopsis and Homo, is around 10%. TargetP also predicts cleavage sites with levels of correctly predicted sites ranging from approximately 40% to 50% (chloroplastic and mitochondrial presequences) to above 70% (secretory signal peptides). TargetP is available as a web-server at http://www.cbs.dtu.dk/services/TargetP/.
A statistical model is presented for computing probabilities that proteins are present in a sample on the basis of peptides assigned to tandem mass (MS/MS) spectra acquired from a proteolytic digest of the sample. Peptides that correspond to more than a single protein in the sequence database are apportioned among all corresponding proteins, and a minimal protein list sufficient to account for the observed peptide assignments is derived using the expectation-maximization algorithm. Using peptide assignments to spectra generated from a sample of 18 purified proteins, as well as complex H. influenzae and Halobacterium samples, the model is shown to produce probabilities that are accurate and have high power to discriminate correct from incorrect protein identifications. This method allows filtering of large-scale proteomics data sets with predictable sensitivity and false positive identification error rates. Fast, consistent, and transparent, it provides a standard for publishing large-scale protein identification data sets in the literature and for comparing the results obtained from different experiments.
All living organisms, no matter how simple or complex, possess the ability to translocate proteins across biological membranes and into different cellular compartments. Although a range of membrane transport processes exist, the major pathway used to translocate proteins across the bacterial cytoplasmic membrane or the eukaryotic endoplasmic reticulum membrane is conserved and is known as the Sec or Sec61 pathway, respectively. Over the past two decades the Sec and Sec61 pathways have been studied extensively and are well characterised at the genetic and biochemical levels. However, it is only now with the recent structural determination of a number of the key elements of the pathways that the translocation complex is beginning to give up its secrets in exquisite molecular detail. This article will focus on the routes of Sec- and Sec61-dependent membrane targeting and the nature of the translocation channel in bacteria and eukaryotes.
Wheat leaf rust is caused by the fungus Puccinia triticina. The genetics of resistance follows the gene-for-gene hypothesis, and thus the presence or absence of a single host resistance gene renders a plant resistant or susceptible to a leaf rust race bearing the corresponding avirulence gene. To investigate some of the changes in the proteomes of both host and pathogen during disease development, a susceptible line of wheat infected with a virulent race of leaf rust were compared to mock-inoculated wheat using 2-DE (with IEF pH 4-8) and MS. Up-regulated protein spots were excised and analyzed by MALDI-QqTOF MS/MS, followed by cross-species protein identification. Where possible MS/MS spectra were matched to homologous proteins in the NCBI database or to fungal ESTs encoding putative proteins. Searching was done using the MASCOT search engine. Remaining unmatched spectra were then sequenced de novo and queried against the NCBInr database using the BLAST and MS BLAST tools. A total of 32 consistently up-regulated proteins were examined from the gels representing the 9-day post-infection proteome in susceptible plants. Of these 7 are host proteins, 22 are fungal proteins of known or hypothetical function and 3 are unknown proteins of putative fungal origin.
Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat adaptation
- J Jia
- S Zhao
- X Kong
- Y Li
Jia, J., Zhao, S., Kong, X., Li, Y. et al., Aegilops tauschii draft
genome sequence reveals a gene repertoire for wheat adaptation. Nature 2013, 496, 91-95.
Rust haustoria: nutrient uptake and beyond
- R T Ogele
- K Mengden
[9] V ¨
ogele, R. T., Mengden, K., Rust haustoria: nutrient uptake
and beyond. New Phytol. 2003, 159, 93–100.
- L V Bindschedler
- T V Burgis
- D J S Mills
- J T C Ho
Bindschedler, L. V., Burgis, T. V., Mills, D. J. S., Ho, J. T.
C. et al., In Planta proteomics and proteogenomics of the
biotrophic barley fungal pathogen Blumeria graminis f. sp.
hordei. Mol. Cell. Proteomics 2009, 8, 2368-2381.
Production and diffusion of chloroplastic H 2 O 2 and its implication to signalling
- M Mubarakshina
- B N Ivanov
- I A Naydov
- W Hilier
Mubarakshina, M., Ivanov, B. N., Naydov, I. A., Hilier, W.
et al., Production and diffusion of chloroplastic H 2 O 2 and its
implication to signalling. J. Exp. Bot. 2010, 61, 3577-3587.
Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat adaptation
- Jia
Sec-dependent protein translocation across biological membranes: evolutionary conservation of an essential protein transport pathway (review)
- Stephenson