![Phosphorylation of Mpk1 T-loop is required for in vivo activity.
(A) Time course of Mpk1 phosphorylation in response to Calcofluor White (CFW), Chlorpromazine (CPZ) and Caffeine (Caff). Mid log phase cells of a strain carrying a HA-tagged Mpk1 allele (UMA44) were treated with the indicated amount of cell wall stressors and aliquots of the same culture were withdrawn before (0) and at the indicated times (in minutes) following compound addition. Protein extracts were prepared and a similar amount of total protein of each time point were loaded in SDS-PAGE gels and submitted to Western blot assays. The same blot, after stripping, was incubated with anti-pTEpY to detect T-loop phosphorylation and with anti-HA to detect HA-tagged proteins. (B) T-loop phosphorylation of Mpk1 in vivo is dependent on upstream kinases. Cell extracts from CFW-treated cultures over a 2 h period were prepared from UMA44 (wt control), UMA44.2 (bck1Δ), UMA44.3 (mkk1Δ), and UMA3 (mpk1Δ), cells and analyzed as described above. (C) Catalytic activity of Mpk1 is enhanced after CFW treatment. HA-tagged proteins were immunoprecipitated from cells extracts prepared from UMA44 cells grown in the presence or absence of 50 µM CFW for 2 h. Protein kinase activity was measured by incubation of immunoprecipitates with purified Myelin Basic Protein (MBP) as substrate and [γ-32P] ATP. On the bottom, 8% SDS-PAGE and immunoblot with anti-HA was used to show comparable levels of Mpk1 proteins in the reaction mixtures. On the top, 12.5% SDS-PAGE and autorradiography was used to detect in vitro phosphorylated MBP. (D) Scheme of Mpk1 showing the mutated residues in Mpk1AEF and Mpk1KD alleles. (E) T-loop phosphorylation of Mpk1 mutants. Cell extracts from CFW-treated cultures over 2 h period were prepared from UMA44 (Mpk1-HA), UMA59 (Mpk1AEF-HA), and UMA60 (Mpk1KD-HA), cells and analyzed as described above. (F) Kinase activity of Mpk1 mutants. Immunoprecipitates from UMA44 (mpk1-HA), UMA3 (mpk1Δ), UMA59 (mpk1AEF-HA), and UMA60 (mpk1KD-HA) cells were analyzed as described above. (G) Sensitivity to CFW of Mpk1 mutants.](https://www.researchgate.net/profile/Jose-Perez-Martin-3/publication/45114713/figure/fig17/AS:341211601752071@1458362593546/Phosphorylation-of-Mpk1-T-loop-is-required-for-in-vivo-activity-A-Time-course-of-Mpk1_Q320.jpg)
![Phosphorylation of Mpk1 T-loop is required for in vivo activity. (A) Time course of Mpk1 phosphorylation in response to Calcofluor White (CFW), Chlorpromazine (CPZ) and Caffeine (Caff). Mid log phase cells of a strain carrying a HA-tagged Mpk1 allele (UMA44) were treated with the indicated amount of cell wall stressors and aliquots of the same culture were withdrawn before (0) and at the indicated times (in minutes) following compound addition. Protein extracts were prepared and a similar amount of total protein of each time point were loaded in SDS-PAGE gels and submitted to Western blot assays. The same blot, after stripping, was incubated with anti-pTEpY to detect T-loop phosphorylation and with anti- HA to detect HA-tagged proteins. (B) T-loop phosphorylation of Mpk1 in vivo is dependent on upstream kinases. Cell extracts from CFW-treated cultures over a 2 h period were prepared from UMA44 (wt control), UMA44.2 ( bck1 D ), UMA44.3 ( mkk1 D ), and UMA3 ( mpk1 D ), cells and analyzed as described above. (C) Catalytic activity of Mpk1 is enhanced after CFW treatment. HA-tagged proteins were immunoprecipitated from cells extracts prepared from UMA44 cells grown in the presence or absence of 50 m M CFW for 2 h. Protein kinase activity was measured by incubation of immunoprecipitates with purified Myelin Basic Protein (MBP) as substrate and [ c - 32 P] ATP. On the bottom, 8% SDS-PAGE and immunoblot with anti-](https://www.researchgate.net/profile/Jose-Perez-Martin-3/publication/45114713/figure/fig2/AS:307368463224833@1450293760401/Phosphorylation-of-Mpk1-T-loop-is-required-for-in-vivo-activity-A-Time-course-of-Mpk1_Q320.jpg)
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Activation of the Cell Wall Integrity Pathway Promotes Escape from G2 in the Fungus Ustilago maydis
PLOS Genetics
Abstract and Figures
It is widely accepted that MAPK activation in budding and fission yeasts is often associated with negative effects on cell cycle progression, resulting in delay or arrest at a specific stage in the cell cycle, thereby enabling cells to adapt to changing environmental conditions. For instance, activation of the Cell Wall Integrity (CWI) pathway in the budding yeast Saccharomyces cerevisiae signals an increase in CDK inhibitory phosphorylation, which leads cells to remain in the G2 phase. Here we characterized the CWI pathway of Ustilago maydis, a fungus evolutionarily distant from budding and fission yeasts, and show that activation of the CWI pathway forces cells to escape from G2 phase. In spite of these disparate cell cycle responses in S. cerevisiae and U. maydis, the CWI pathway in both organisms appears to respond to the same class cell wall stressors. To understand the basis of such a difference, we studied the mechanism behind the U. maydis response. We found that activation of CWI pathway in U. maydis results in a decrease in CDK inhibitory phosphorylation, which depends on the mitotic phosphatase Cdc25. Moreover, in response to activation of the CWI pathway, Cdc25 accumulates in the nucleus, providing a likely explanation for the increase in the unphosphorylated form of CDK. We also found that the extended N-terminal domain of Cdc25, which is dispensable under normal growth conditions, is required for this G2 escape as well as for resistance to cell wall stressors. We propose that the process of cell cycle adaptation to cell stress evolved differently in these two divergent organisms so that each can move towards a cell cycle phase most appropriate for responding to the environmental signals encountered.
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Supplementary resources (6)
... As shown in Figure 7, the U. maydis mutant (in contrast to the S. cerevisiae ppz1 mutant) did not display altered tolerance to NaCl or LiCl, and was not sensitive to heat or oxidative stress (H2O2). U. maydis cells deficient in UmPpz1 also displayed normal tolerance to calcium ions or chlorpromazine, an activator of Cell Wall Integrity (CWI) pathway in this fungus [34] (not shown). In contrast, the mutant was The empty pGEX6P-1 plasmid (∅) and the same vector bearing the Arabidopsis AtHal3 (as positive control), ScHal3 (as negative control), full-length UmHal3, and the indicated UmHal3 variants were introduced into the E. coli strain BW369, which carries the dfp-707ts mutation that abolish PPCDC activity when cells are grown at 37 • C. Transformants were grown at 30 • C, diluted till OD 600 = 0.05, transferred to the permissive (30 • C) or 37 • C (non-permissive) temperatures for 6 h, and then plated at the indicated temperatures. ...
... As shown in Figure 7, the U. maydis mutant (in contrast to the S. cerevisiae ppz1 mutant) did not display altered tolerance to NaCl or LiCl, and was not sensitive to heat or oxidative stress (H 2 O 2 ). U. maydis cells deficient in UmPpz1 also displayed normal tolerance to calcium ions or chlorpromazine, an activator of Cell Wall Integrity (CWI) pathway in this fungus [34] (not shown). In contrast, the mutant was more sensitive to caffeine and calcofluor white, two compounds known to interfere with normal cell wall construction. ...
... UmEna1 is a typical ENA ATPase, although it shows low-Na + /K + discrimination, whereas UmEna2 shows a different endosomal/plasma membrane distribution, and its function is still unclear [39]. In addition, it has been demonstrated that U. maydis escapes from G 2 phase by activating the CWI pathway [34], which contrasts with the G 2 cell cycle arrest that occurs in S. cerevisiae in response to CWI pathway activation [40,41]. Therefore, even if the phosphatases in both organisms are functionally related to the CWI, the underlying mechanisms are likely different. ...
Ppz enzymes are type-1 related Ser/Thr protein phosphatases that are restricted to fungi. In S. cerevisiae and other fungi, Ppz1 is involved in cation homeostasis and is regulated by two structurally-related inhibitory subunits, Hal3 and Vhs3, with Hal3 being the most physiologically relevant. Remarkably, Hal3 and Vhs3 have moonlighting properties, as they participate in an atypical heterotrimeric phosphopantothenoyl cysteine decarboxylase (PPCDC), a key enzyme for Coenzyme A biosynthesis. Here we identify and functionally characterize Ppz1 phosphatase (UmPpz1) and its presumed regulatory subunit (UmHal3) in the plant pathogen fungus Ustilago maydis. UmPpz1 is not an essential protein in U. maydis and, although possibly related to the cell wall integrity pathway, is not involved in monovalent cation homeostasis. The expression of UmPpz1 in S. cerevisiae Ppz1-deficient cells partially mimics the functions of the endogenous enzyme. In contrast to what was found in C. albicans and A. fumigatus, UmPpz1 is not a virulence determinant. UmHal3, an unusually large protein, is the only functional PPCDC in U. maydis and, therefore, an essential protein. However, when overexpressed in U. maydis or S. cerevisiae, UmHal3 does not reproduce Ppz1-inhibitory phenotypes. Indeed, UmHal3 does not inhibit UmPpz1 in vitro (although ScHal3 does). Therefore, UmHal3 might not be a moonlighting protein.
... As a first approach to this idea, we have performed mass spectrometry analysis of a functional Sir2:eGFP ( Figure 7A) pull down and found, among others, a transcription factor involved in virulence, Med1 (Chacko and Gold, 2012), to interact with Sir2. Additional interesting interactors found in our analysis are the mitogen-activated protein kinase (MAPK) Mkk1 involved in cell wall integrity signaling (Carbó and Pérez-Martín, 2010) and five uncharacterized proteins ( Figure 7B). ...
Phytopathogenic fungi must adapt to the different environmental conditions found during infection and avoid the immune response of the plant. For these adaptations, fungi must tightly control gene expression, allowing sequential changes in transcriptional programs. In addition to transcription factors, chromatin modification is used by eukaryotic cells as a different layer of transcriptional control. Specifically, the acetylation of histones is one of the chromatin modifications with a strong impact on gene expression. Hyperacetylated regions usually correlate with high transcription and hypoacetylated areas with low transcription. Thus, histone deacetylases (HDACs) commonly act as repressors of transcription. One member of the family of HDACs is represented by sirtuins, which are deacetylases dependent on NAD+, and, thus, their activity is considered to be related to the physiological stage of the cells. This property makes sirtuins good regulators during environmental changes. However, only a few examples exist, and with differences in the extent of the implication of the role of sirtuins during fungal phytopathogenesis. In this work, we have performed a systematic study of sirtuins in the maize pathogen Ustilago maydis , finding Sir2 to be involved in the dimorphic switch from yeast cell to filament and pathogenic development. Specifically, the deletion of sir2 promotes filamentation, whereas its overexpression highly reduces tumor formation in the plant. Moreover, transcriptomic analysis revealed that Sir2 represses genes that are expressed during biotrophism development. Interestingly, our results suggest that this repressive effect is not through histone deacetylation, indicating a different target of Sir2 in this fungus.
... In S. cerevisiae, cell stressors, such as CR and Caspofungin, are sensed by the Mid2 and Wsc1 sensors (Jin et al. 2013). Similar sensors appear to be involved in sensing these cell wall stressors in fungal pathogens, such as U. maydis and F. graminearum (Carbó and Pérez-Martín 2010;Xu et al. 2019). In yeast, cell wall damage caused by β-1,3-glucanase and protease activities is sensed by Sho1 and mucin Hkr1 (not Msb2) to activate Hog1, which in turn activates Slt2 (Rodríguez-Peña et al. 2013). ...
Like other eukaryotes, fungi use MAP kinase (MAPK) pathways to mediate cellular changes responding to external stimuli. In the past two decades, three well-conserved MAP kinase pathways have been characterized in various plant pathogenic fungi for regulating responses and adaptations to a variety of biotic and abiotic stresses encountered during plant infection or survival in nature. The invasive growth (IG) pathway is homologous to the yeast pheromone response and filamentation pathways. In plant pathogens, the IG pathway often is essential for pathogenesis by regulating infection-related morphogenesis, such as appressorium formation, penetration, and invasive growth. The cell wall integrity (CWI) pathway also is important for plant infection although the infection processes it regulates vary among fungal pathogens. Besides its universal function in cell wall integrity, it often plays a minor role in responses to oxidative and cell wall stresses. Both the IG and CWI pathways are involved in regulating known virulence factors as well as effector genes during plant infection and mediating defenses against mycoviruses, bacteria, and other fungi. In contrast, the high osmolarity growth (HOG) pathway is dispensable for virulence in some fungi although it is essential for plant infection in others. It regulates osmoregulation in hyphae and is dispensable for appressorium turgor generation. The HOG pathway also plays a major role for responding to oxidative, heat, and other environmental stresses and is overstimulated by phenylpyrrole fungicides. Moreover, these three MAPK pathways crosstalk and coordinately regulate responses to various biotic and abiotic stresses. The IG and CWI pathways, particularly the latter, also are involved in responding to abiotic stresses to various degrees in different fungal pathogens, and the HOG pathway also plays a role in interactions with other microbes or fungi. Furthermore, some infection processes or stress responses are co-regulated by MAPK pathways with cAMP or Ca ²⁺ /CaM signaling. Overall, functions of individual MAP kinase pathways in pathogenesis and stress responses have been well characterized in a number of fungal pathogens, showing the conserved genetic elements with diverged functions, likely by rewiring transcriptional regulatory networks. In the near future, applications of genomics and proteomics approaches will likely lead to better understanding of crosstalk among the MAPKs and with other signaling pathways as well as roles of MAPKs in defense against other microbes (biotic interactions).
... In fungi, morphology and the cell cycle are intricately connected (Sartorel and Perez-Martin, 2012). Fungi impose delays or arrests at specific cell cycle stages to enable the cell to adapt to unfavorable stress conditions or to synchronize cell cycle progression before mating, which is negatively regulated by MAPK cascades (Carbó and Pérez-Martín, 2010). In response to pheromone recognition, cell cycle arrest regulated by MAPK signaling occurs in the process of budding, leading to the formation of conjugation tubes before cell fusion (Garcia-Muse et al., 2003). ...
Ustilago esculenta undergoes an endophytic life cycle in Zizania latifolia. It induces the stem of its host to swell, forming the edible galls called jiaobai in China, which are the second most commonly cultivated aquatic vegetable in China. Z. latifolia raised for jiaobai can only reproduce asexually because the U. esculenta infection completely inhibits flowering. The infection and proliferation in the host plants during the formation of edible gall differ from those of conventional pathogens. Previous studies have shown a close relationship between mitogen-activated protein kinase (MAPK) and fungal pathogenesis. In this study, we explored the functional properties of the MAPK UeKpp2. Cross-species complementation assays were carried out, which indicated a functional complementation between the UeKpp2 of U. esculenta and the Kpp2 of Ustilago maydis. Next, UeKpp2 mutants of the UeT14 and the UeT55 sporidia background were generated; these showed an aberrant morphology of budding cells, and attenuated mating and filamentous growth in vitro, in the context of normal pathogenicity. Interestingly, we identified another protein kinase, UeUkc1, which acted downstream of UeKpp2 and may participate in the regulation of cell shape. We also found a defect of filamentous growth in UeKpp2 mutants that was not related to a defect of the induction of mating-type genes but was directly related to a defect in UeRbf1 induction. Overall, our results indicate an important role for UeKpp2 in U. esculenta that is slightly different from those reported for other smut fungi.
... It has been reported that CFW and CR perturb cell wall assembly by binding to chitin. SDS is known to act indirectly by perturbation of the plasma membrane, resulting in cell wall debilitation and triggering stress responses [38][39][40]. Based on the literature, it is Fig. 6. ...
Fusarium verticillioides is one of the key fungal pathogens responsible for maize stalk rot. While stalk rot pathogens are prevalent worldwide, our understanding of the stalk rot virulence mechanism in pathogenic fungi is still very limited. We previously identified the F. verticillioides FvSYN1 gene, which was demonstrated to play an important role in maize stalk rot virulence. FvSyn1 belongs to a family of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that play critical roles in a variety of developmental processes. In this study, we further characterized the cellular features of the FvSyn1 protein, namely how different motifs contribute to development and virulence in F. verticillioides by generating motif-specific deletion mutants. Microscopic observation showed that the ∆Fvsyn1 mutant exhibits rough and hyper-branched hyphae when compared to the wild-type progenitor. Moreover, the ∆Fvsyn1 mutant was sensitive to cell wall stress agents, resulting in vegetative growth reduction. We showed that the FvSyn1::GFP protein is associated with the endomembrane, but this did not clarify why the deletion of FvSyn1 led to stress sensitivity and aberrant hyphal development. Characterization of the FvSyn1 domains indicated that both the syntaxin N-terminus (SynN) domain and the SNARE C-terminus domain play distinct roles in fungal development, but also function collectively in the context of virulence. We also determined that two domains in FvSyn1 are not required for fumonisin production. Interestingly, these two domains were involved in carbon nutrient utilization, including pectin, starch and sorbitol. This study further characterized the role of FvSyn1 domains in hyphal growth, cell wall stress response and virulence in F. verticillioides.
The fungal cell wall is the first layer exposed to the external environment. The cell wall has key roles in regulating cell functions, such as cellular stability, permeability, and protection against stress. Understanding the structure of the cell wall and the mechanism of its biogenesis is important for the study of fungi. Highly conserved in fungi, including Magnaporthe oryzae, the cell wall–integrity (CWI) pathway is the primary signaling cascade regulating cell-wall structure and function. The CWI pathway has been demonstrated to correlate with pathogenicity in many phytopathogenic fungi. In the synthesis of the cell wall, the CWI pathway cooperates with multiple signaling pathways to regulate cell morphogenesis and secondary metabolism. Many questions have arisen regarding the cooperation of different signaling pathways with the CWI pathway in regulating cell-wall synthesis and pathogenicity. In this review, we summarized the latest advances in the M. oryzae CWI pathway and cell-wall structure. We discussed the CWI pathway components and their involvement in different aspects, such as virulence factors, the possibility of the pathway as a target for antifungal therapies, and crosstalk with other signaling pathways. This information will aid in better understanding the universal functions of the CWI pathway in regulating cell-wall synthesis and pathogenicity in M. oryzae.
[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
Crucifers’ host-pathosystem has become a favorable model system of biologists to get deeper insight into various biological functions operating by induction of differential gene expressions. The genomics of crucifers’ host-pathosystem has been strengthened with different omics approaches/techniques used to reveal genome sequences of major Brassica crops and their pathogens. Some of the selected techniques/protocols developed and used by different scientists have been included in this chapter for the benefit of Brassica teachers, researchers, and students. The isolates of A. candida collected from field have been characterized with the use of DNA sequence approaches. A PCR test specific only to the presence of A. candida DNA has been developed which allows separation of host DNA and detection of pathogen from both symptomatic and asymptomatic leaf material as well as DNA from plant species for which Albugo infection is unknown. The protocol for genome sequencing and assembly of 16 Alternaria species with mRNA sequencing and transcript construction has been developed. The real time PCR based method has been used for quantitative estimation of seed infection of Brassica by A. brassicae. The Brassica–Alternaria interactions have been analyzed with microarray approach. A procedure has been detailed for molecular and biochemical changes using Brassica–Alternaria host-pathosystem. A detailed method has been developed for assessing virulence variability of Alternaria species infecting crucifer’s. With the Brassica–Colletotrichum host-pathosystem, expression of MAPK genes has been characterized. A molecular identification technique for Erysiphe at anamorphic stage in conjunction with morphological and host data is very valuable for quick results. A procedure has been used to identify powdery mildew isolates through DNA sequence analysis and DNA markers analysis. Histological assessment of Erysiphe growth in host tissues has been revealed. A fast molecular marker identification of Hyaloperonospora method based on multiplex PCR (rDNA) analysis is useful for diagnosis of crucifers’ downy mildew. The DNA finger printing with RAPD analysis can distinguish H. parasitica isolates from Brassica species. A method for DNA isolation from infected hosts by H. parasitica has been developed. A method has been developed to characterize Leptosphaeria isolates for virulence on different Brassica species. The Leptosphaeria maculans field populations have been analyzed for genetic diversity. Phylogenetic relationship of R-loci in Brassica species to Leptosphaeria has been determined through DNA-PCR analysis. A protocol has been developed for transcriptome analysis of Brassica–Leptosphaeria pathosystem to reveal molecular mechanisms. The PCR based protocols have been developed to detect P. brassicae from soil, and plant along with its quantification. Bait plants have been used to validate the real time PCR assay method. The Plasmodiophora pathotype-specific genes have been identified with PCR and qPCR approach. The qPCR approach has been used for P. brassicae quantification from soil to assess response of time based fertilizers and other control measures along with its spread. The RNA seq analysis method has been developed for assessing genes expression during Plasmodiophora interactions with their host plants. The Plasmodiophora brassicae genome has been sequenced along with transcriptomic analysis during B. napus infection. The approach of transcriptomic analysis of R and S genotypes of Brassica during Brassica–Sclerotinia interaction has revealed genes expression controlling resistance and susceptibility of host plants. A serological test has been developed that allows the early detection of rapeseed petal infection by Sclerotinia inoculum under field conditions which has potential to visualize risk of infection. The infection of different viruses in Brassica has been detected with multiplex RT-PCR approach. The qRT-PCR assay has been used to detect expression levels of BnSGS3 gene in different tissues of Brassica nontransgenic, single transgenic, and virus inoculated plants. The presence of viruses on crucifers has been detected with both biological and serological methods. The isolates of Verticillium collected from different hosts and locations have been characterized with molecular PCR technique. Protocol has been developed to determine defensive metabolites in the phenylpropanoid pathways during Brassica–Xanthomonas interactions. A molecular method has been developed for genotyping of Xanthomonas campestris pv. campestris isolates. Multispectral imaging approach has been developed for presymptomatic identification of crucifers’ pathogens/disease. The RAPD-PCR approach has been used for the diagnosis of mixed infection in Brassica crops.KeywordsProtocols for host-pathosystems studiesAlbugo techniquesUse of DNA sequence approachPCR assay for detectionAlternaria techniquesGenome sequencingPCR-based assay for detectionMicroarray analysisMolecular and biochemical analysisVirulence variabilityColletotrichum techniquesCharacterization of MAPK geneErysiphe techniquesMolecular identificationDNA sequence analysisDNA marker analysisHistological assessmentHyaloperonospora techniquesMolecular marker for identificationDNA finger printingDNA isolationLeptosphaeria techniquesCharacterization of isolatesGenetic variabilityPhylogenetic relationship of R-lociTranscriptome analysisPlasmodiophora techniquesPCR detectionPCR technique to identify pathogenReal time PCR assayIdentification of pathotype-specific genesQuantification and assessment with qPCRPCR and quantitative PCR analysisGenome sequencing and transcriptomic analysisSclerotinia techniquesTranscriptome analysisImmunoassay for early detectionTuMV techniquesDetection by multiplex reverse transcription polymerase chain reactionRT-PCR assay for detection of BnSGS3 geneVerticillium techniquesMolecular characterizationXanthomonas techniquesDetermination of defensive metabolitesGenotyping of XanthomonasMixed infection study techniquesMultispectral imagingRAPD-based molecular diagnosis
Plant pathogenic fungi use the well-conserved MAP kinase (MAPK) pathways to mediate responses to external stimuli and regulate various infection and developmental processes. Most ascomycetous fungal pathogens have three MAPK cascades. In general, the Pmk1/Kss1 invasive growth (IG) pathway is essential for pathogenesis by regulating infection-related morphogenesis, such as formation of appressoria or hyphopodia, penetration, and invasive growth in infected plant tissues. The cell wall integrity (CWI) MAPK pathway is also normally important for plant infection by regulating species-specific infection processes, cell wall integrity, infectious growth, and responses to cell wall stress. Unlike the IG and CWI pathways, the HOG (high osmolarity glycerol) pathway is dispensable for virulence in some fungal pathogens such as Magnaporthe oryzae but plays a critical role in pathogenesis in many others. Besides its conserved role in osmoregulation, the HOG pathway is usually important for responses to oxidative and other environmental stresses. Overall, both conserved and species-specific functions have been identified for individual MAP kinase cascades in plant pathogenic fungi, likely due to variations in upstream signaling and downstream transcriptional regulation. Limited studies in a few fungal pathogens have also shown that there is crosstalk among three MAPK pathways to regulate various infection processes and responses to biotic and abiotic stresses, indicating the complex regulatory networks associated with these MAP kinase pathways.KeywordsSignal transductionPathogenesis
PMK1
Appressorium formationPlant penetrationCell wall integrity
The pathogenic fungus Phomopsis longicolla causes numerous plant diseases, such as Phomopsis seed decay, pod and stem blight, and stem canker, which seriously affect the yield and quality of soybean production worldwide. Because of a lack of technology for efficient manipulation of genes for functional genomics, understanding of P. longicolla pathogenesis is limited. Here, we developed an efficient polyethylene glycol–mediated protoplast transformation system in P. longicolla that we used to characterize the functions of two genes involved in the cell wall integrity (CWI) pathway of the mitogen-activated protein kinase (MAPK) cascade, including PlMkk1, which encodes MAPK kinase, and its downstream gene PlSlt2, which encodes MAPK. Both gene knockout mutants ΔPlMkk1 and ΔPlSlt2 displayed a reduced growth rate, fragile aerial hyphae, abnormal polarized growth and pigmentation, defects in sporulation, inadequate CWI, enhanced sensitivity to abiotic stress agents, and significant deficiencies in virulence, although there were some differences in degree. The results suggest that PlMkk1 and PlSlt2 are crucial for a series of growth and development processes as well as pathogenicity. The developed transformation system will be a useful tool for additional gene function research and will aid in the elucidation of the pathogenic mechanisms of P. longicolla.
[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
In the fungus Ustilago maydis, sexual pheromones elicit mating resulting in an infective filament able to infect corn plants. Along this process a G2 cell cycle arrest is mandatory. Such as cell cycle arrest is initiated upon the pheromone recognition in each mating partner, and sustained once cell fusion occurred until the fungus enter the plant tissue. We describe that the initial cell cycle arrest resulted from inhibition of the nuclear transport of the mitotic inducer Cdc25 by targeting its importin, Kap123. Near cell fusion to take place, the increase on pheromone signaling promotes Cdc25 degradation, which seems to be important to ensure the maintenance of the G2 cell cycle arrest to lead the formation of the infective filament. This way, premating cell cycle arrest is linked to the subsequent steps required for establishment of the infection. Disabling this connection resulted in the inability of fungal cells to infect plants.
A wide diversity of treatments can cause rapid and extensive bursting of hyphal tips of Mucor rouxii. Most hyphal tips from colonies grown on full-strength agar medium burst readily when flooded with distilled water. In contrast, hyphal tips from colonies grown on diluted medium survived flooding with distilled water but succumbed to dilute aqueous solutions (particularly acids, but also neutral salts, EDTA, alcohols, acetone, detergents). Apex bursting was generally inhibited by alkaline solutions but took place at certain concentrations of ethanolamine or NH40OH. Mg2+, Mn2+ or Ca2+ caused pronounced swelling of the hyphal apex followed by an occasional burst. Sharp temperature increases also brought about apical disintegration regardless of nutrient concentration.
Colonies of Aspergillus fumigatus, Fusarium sp., Neurospora crassa (wild-type and two morphological mutants), Penicillium claviforme, Rhizoctonia solani and Rhizopus arrhizus also underwent apical bursting when flooded with water or rapidly heated. Schizophyllum commune, several Phytophthora spp., and some morphological mutants of N. crassa were refractory.
The bursting of hyphal tips by flooding colonies with water is not simply an osmotic phenomenon; its temperature coefficient (Q10 1.3 to 2.0) suggests the additional participation of biochemical reaction(s) as a rate-limiting step in the bursting process. Probably, enzymatic degradation and hence weakening of the wall is a prerequisite for bursting. These observations are offered as circumstantial evidence supporting the following conclusions: (i) the growing tips of fungi have a large wall lytic potential; (ii) the release of this activity during growth must be gradual and delicately co-ordinated with wall synthesis; (iii) this balance can be easily disturbed by a wide variety of external stimuli and the ensuing surge of lytic activity results in the violent disintegration of the hyphal apex.
Mitogen-activated protein kinase (MAPK) signaling pathways are critical for the sensing and response of eukaryotic cells to
extracellular changes. In Schizosaccharomyces pombe, MAPK Pmk1/Spm1 has been involved in cell wall construction, morphogenesis, cytokinesis, and ion homeostasis, as part of
the so-called cell integrity pathway together with MAPK kinase kinase Mkh1 and MAPK kinase Pek1. We show that Pmk1 is activated
in multiple stress situations, including hyper- or hypotonic stress, glucose deprivation, presence of cell wall-damaging compounds,
and oxidative stress induced by hydrogen peroxide or pro-oxidants. The stress-induced activation of Pmk1 was completely dependent
on Mkh1 and Pek1 function, supporting a nonbranched pathway in the regulation of MAPK activation. Fluorescence microscopy
revealed that Mkh1, Pek1, and Pmp1 (a protein phosphatase that inactivates Pmk1) are cytoplasmic proteins. Mkh1 and Pek1 were
also found at the septum, whereas Pmk1 localized in both cytoplasm and nucleus as well as in the mitotic spindle and septum
during cytokinesis. Interestingly, Pmk1 subcellular localization was unaffected by stress or the absence of Mkh1 and Pek1,
suggesting that its activation by the Mkh1-Pek1 cascade takes place at the cytoplasm and/or septum and that the active and
inactive forms of this kinase cross the nuclear membrane. Cdc42 GTPase and its effectors, p21-activated kinases Pak2 and Pak1,
are not upstream elements controlling the basal level or the stress-induced activation of Pmk1. However, Sty1 MAPK was essential
for proper Pmk1 deactivation after hypertonic stress in a process regulated by Atf1 transcription factor. These results provide
the first evidence for the existence of cross-talk between two MAPK cascades during the stress response in fission yeast.
Signal transduction mediated by the mitogen-activated protein kinase (MAPK) Slt2 pathway is essential to maintain the cell wall integrity in Saccharomyces cerevisiae. Stimulation of MAPK pathways results in activation by phosphorylation of conserved threonine and tyrosine residues of MAPKs. We have used an antibody that specifically recognizes dually phosphorylated Slt2 to gain insight into the activation and modulation of signaling through the cell integrity pathway. We show that caffeine and vanadate activate this pathway in the absence of osmotic stabilization. The lack of the putative cell surface sensor Mid2 prevents vanadate- but not caffeine-induced Slt2 phosphorylation. Disruption of the Rho1-GTPase-activating protein genes SAC7 and BEM2 leads to constitutive Slt2 activation, indicating their involvement as negative regulators of the pathway. MAPK kinases also seem to participate in signaling regulation, Mkk1 playing a greater role than Mkk2 in signal transmission to Slt2. Additionally, one of the phosphatases involved in Slt2 dephosphorylation is likely to be the dual specificity phosphatase Msg5, since overexpression of MSG5 in a sac7Delta mutant eliminates the high Slt2 phosphorylation, and disruption of MSG5 in wild type cells results in increased phospho-Slt2 levels. These data present the first evidence for a negative regulation of the cell integrity pathway.
During induction of the virulence program in the phytopathogenic fungus Ustilago maydis, the cell cycle is arrested on the plant surface and it is not resumed until the fungus enters the plant. The mechanism of this cell cycle arrest is unknown, but it is thought that it is necessary for the correct implementation of the virulence program. Here, we show that this arrest takes place in the G2 phase, as a result of an increase in the inhibitory phosphorylation of the catalytic subunit of the mitotic cyclin-dependent kinase Cdk1. Sequestration in the cytoplasm of the Cdc25 phosphatase seems to be one of the reasons for the increase in inhibitory phosphorylation. Strikingly, we also report the DNA-damage checkpoint kinase Chk1 appears to be involved in this process. Our results support the emerging idea that checkpoint kinases have roles other than in the DNA-damage response, by virtue of their ability to interact with the cell cycle machinery.
In Ustilago maydis, cAMP signalling is crucial for successful infection of maize plants. Strains are non-pathogenic if mutated in any of the currently identified components of this signalling pathway, such as the α-subunit of a heterotrimeric G protein Gpa3, the adenylate cyclase Uac1 and the regulatory and catalytic subunit of protein kinase A Ubc1 and Adr1 respectively. Deletion of gpa3, uac1 or adr1 triggers filamentous growth, and certain point mutations in gpa3 and ubc1 that mimic a high cAMP level display a glossy colony phenotype. Screening an autonomously replicating U. maydis library in such a background (gpa3Q206L), we identified sql1 as a suppressor of the glossy colony phenotype. Interestingly, only alleles carrying C-terminal truncations of Sql1 were able to complement the mutant phenotype, suggesting a gain-of-function by these variants. Sql1 is a functional homologue of the yeast transcriptional repressor Ssn6p and contains 10 tetratricopeptide repeats (TPRs), of which the first six are important for suppressor function. Truncated sql1 alleles that suppress the glossy colony phenotype of gpa3Q206L strains induce filamentous growth when introduced in wild type. Filamentation of these strains is reversed in the presence of cAMP. We present a model in which Sql1 is part of an evolutionary conserved Sql1–Tup1 transcriptional repressor complex that antagonizes cAMP signalling by repressing cAMP-regulated genes.
The ability to switch between a yeast-like form and a filamentous form is an extended characteristic among several fungi. In pathogenic fungi, this capacity has been correlated with virulence because along the infection process, dimorphic transitions are often required. Two well-known organisms for which dimorphism have been studied are the pathogenic fungi Candida albicans and Ustilago maydis, which infect mammals and corn, respectively. In both cases, several signal transduction pathways have been defined. Not surprisingly, these pathways are similar to the well-known pathways involved in the pseudohyphal differentiation that some Saccharomyces cerevisiae diploid strains show when nutrients are starved. However, in spite of similarities at the molecular level, strikingly, fungi use similar pathways to respond to environmental inputs, but with differing outcomes.
This account reflects diverse aspects of hyphal growth and development that I have encountered since first becoming interested in fungi. Apical growth is discussed, with particular reference to the phenomena of hyphal maturation and the formation of helical hyphae. It is contrasted with cases of intercalary growth and reversed growth of hyphae. Localization of chitin synthesis at the apex and at other particular sites in the wall is described in detail. It is suggested that chitin microfibrils are synthesized at hyphal tips and at other sites by a transmembrane enzyme complex of several polypeptides allosterically regulated by effector molecules but also perhaps by local physical stress of the membrane. Behaviour of hyphae is discussed with examples of mating zygophores, of contact sensing of surfaces and of responses to applied electric fields.
