Identification and Characterization of In planta–Expressed Secreted Effector Proteins from Magnaporthe oryzae That Induce Cell Death in Rice

Abstract

Interactions between rice and Magnaporthe oryzae involve the recognition of cellular components and the exchange of complex molecular signals from both partners. How these interactions occur in rice cells is still elusive. We employed robust-long serial analysis of gene expression (RL-SAGE), massively parallel signature sequencing (MPSS), and sequencing by synthesis (SBS), to examine transcriptome profiles of infected rice leaves. A total of 6,413 in-planta expressed fungal genes including 851 genes encoding predicted effector proteins were identified. We used a protoplast transient expression system to assess 42 of the predicted effector proteins for the ability to induce plant cell death. Ectopic expression assays identified five novel effectors that induced host cell death only when they contained the signal peptide for secretion to the extracellular space. The five apoplastic effectors induced cell death in a non-host-selective-manner. Although the five effectors are highly diverse in their sequences, the physiological basis of cell death induced by each was similar. This study demonstrates that our integrative genomic approach is effective for the identification of in-planta expressed cell death-inducing effectors from M. oryzae that may play an important role facilitating colonization and fungal growth during infection.

Full text

Vol. 26, No. 2, 2013 / 191
MPMI Vol. 26, No. 2, 2013, pp. 191–202. http://dx.doi.org/10.1094/MPMI -05-12-0117-R.
Identification and Characterization
of In planta–Expressed Secreted Effector Proteins
from Magnaporthe oryzae That Induce Cell Death in Rice
Songbiao Chen,1,2,3 Pattavipha Songkumarn,2 R. C. Venu,2 Malali Gowda,2 Maria Bellizzi,2 Jinnan Hu,2
Wende Liu,1 Daniel Ebbole,4 Blake Meyers,5 Thomas Mitchell,2 and Guo-Liang Wang1,2
1State Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of
Agricultural Sciences, Beijing 100193, China; 2Department of Plant Pathology, The Ohio State University, Columbus, OH
43210, U.S.A.; 3Biotechnology Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003, China;
4Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 79016, U.S.A.; 5Delaware
Biotechnology Institute, University of Delaware, Newark, DE, U.S.A.
Submitted 16 May 2012. Accepted 24 September 2012.
Interactions between rice and Magnaporthe oryzae involve
the recognition of cellular components and the exchange of
complex molecular signals from both partners. How these
interactions occur in rice cells is still elusive. We employed
robust-long serial analysis of gene expression, massively
parallel signature sequencing, and sequencing by synthesis
to examine transcriptome profiles of infected rice leaves. A
total of 6,413 in planta–expressed fungal genes, including
851 genes encoding predicted effector proteins, were iden-
tified. We used a protoplast transient expression system to
assess 42 of the predicted effector proteins for the ability to
induce plant cell death. Ectopic expression assays identi-
fied five novel effectors that induced host cell death only
when they contained the signal peptide for secretion to the
extracellular space. Four of them induced cell death in Nico-
tiana benthamiana. Although the five effectors are highly
diverse in their sequences, the physiological basis of cell
death induced by each was similar. This study demonstrates
that our integrative genomic approach is effective for the
identification of in planta–expressed cell death–inducing
effectors from M. oryzae that may play an important role
facilitating colonization and fungal growth during infection.
Coevolution of plants and their pathogens in nature has led
both sides to develop a battery of strategies to attack and de-
fend. Plant pathogens may first use cell wall–degrading enzymes
to digest the surface layers of cell walls to facilitate penetra-
tion. Successful pathogens can secrete a variety of extracellular
molecules to modulate host defense circuitry. These extracellu-
lar molecules include apoplastic effectors such as virulence
factors, toxins, and degradative enzymes that function as key
virulence determinants to suppress host defense (Hogenhout et
al. 2009). On the other hand, plants have evolved unique mecha-
nisms to defend themselves from most microbes using physi-
cal barriers, antimicrobial compounds, and the innate immune
system. The plant immune system consists of two layers
(Chisholm et al. 2006; Jones and Dangl 2006). The first layer
is initiated by the perception of pathogen- or microbe-associ-
ated molecular patterns (PAMPs or MAMPs) by host mem-
brane-associated pattern recognition receptors (PRR), which
cause basal defense responses in plants, called PAMP-trig-
gered immunity (Zhang and Zhou 2010). The second layer is
initiated by rapid activation of a hypersensitive reaction (HR)
upon recognition of the avirulence effectors by the cognate re-
sistance (R) proteins, which causes strong race-specific resis-
tance called effector-triggered immunity.
Over the past decade, extensive studies have led to the iden-
tification of many PAMP and avirulence effectors in plant
pathogens and PRR and R proteins in plants (Dodds et al.
2009). Among them the majority of the well-characterized
effectors are from prokaryotic bacteria that employ a type III
secretion system to deliver effectors directly into host cells.
Unlike bacteria, fungal and oomycete pathogens often secrete
effector proteins to the extracellular milieu through the eukary-
otic secretory pathway (Panstruga and Dodds 2009). Many se-
creted proteins function in the apoplast of plants, and some
have been shown to be translocated into plant cells (Dou et al.
2008a; Whisson et al. 2007), in which they function in the host
cytoplasm (Armstrong et al. 2005; Bos et al. 2006; Catanzariti
et al. 2006; Dodds et al. 2004; Rehmany et al. 2005; Yoshida et
al. 2009). Recent advances in genome sequencing technologies
have led to a rapid discovery of numerous effectors in oomy-
cete pathogens and have provided a wealth of information on
their structure and function. For example, analysis of the Phy-
tophthora sojae and P. r am o r u m genomes led to the discovery
of conserved motifs RXLR and dEER (Tyler et al. 2006), re-
quired for the translocation of oomycete effectors into plant
cells (Dou et al. 2008a; Whisson et al. 2007). The large family
of predicted RXLR proteins thus provided a wealth of candi-
dates for functional studies of effector activities in oomycetes.
However, secreted proteins exported by fungi appear to lack
such conserved motifs, and little is known about their functions.
The hemibiotrophic fungus Magnaporthe oryzae is the causal
agent of rice blast, the most devastating disease that affects
rice production worldwide (Dean et al. 2005; Ebbole 2007;
Talbot 2003). In M. oryzae, a larger number of genes (up to
S. Chen, P. Songkumarn, and R. C. Venu contributed equally to this work.
Corresponding author: Guo-Liang Wang; Department of Plant Pathology;
Ohio State University; Telephone: +1.614.292.9280; Fax: +1.614.292.4455;
E-mail: wang.620@osu.edu
*The e-Xtra logo stands for “electronic extra” and indicates three
supplementary tables, two supplementary figures, and supplementary
methods information are published online.
©
2013 The American Phytopathological Society
e-Xt
r
a*

192 / Molecular Plant-Microbe Interactions
1,306) coding for putative secreted proteins have been pre-
dicted from the genome of a laboratory strain, 70-15 (Dean et
al. 2005; Yoshida et al. 2009). Seven secreted proteins, i.e.,
PWL1, PWL2 (Kang et al. 1995; Sweigard et al. 1995), AvrPi-
ta (Orbach et al. 2000), Avr-Pia, Avr-Pii, Avr-Pik/km/kp
(Yoshida et al. 2009), and AvrPiz-t (Li et al. 2009), have been
confirmed as avirulence (Avr) proteins presumably recognized
by the corresponding resistance gene products. In addition, a
few secreted proteins that are required for pathogenicity, i.e.,
MPG1 (Talbot et al. 1993), EMP1 (Ahn et al. 2004), MHP1
(Kim et al. 2005), MSP1 (Jeong et al. 2007), MC69 (Saitoh et
al. 2012), and Slp1 (Mentlak et al. 2012), and four biotrophy-
associated secreted proteins, BAS1 to BAS4 (Mosquera et al.
2009), have also been characterized. However, the majority of
M. oryzae secreted proteins have not been experimentally
tested for their functions in pathogenicity.
To identify M. oryzae genes encoding predicted secreted
proteins that were expressed in blast-infected leaf tissue, we
developed an integrative genome expression profiling approach
that includes robust-long serial analysis of gene expression
(RL-SAGE) (Gowda et al. 2004), massively parallel signature
sequencing (MPSS) (Brenner et al. 2000; Meyers et al. 2004;
Nobuta et al. 2007), and sequencing by synthesis (SBS)
(German et al. 2008; Venu et al. 2011a). Infected tissues were
collected from six timepoints so that fungal transcripts ex-
pressed in planta at a late infection stage can be included in the
libraries. We used a protoplast transient expression assay to
identify in planta–expressed secreted proteins from M. oryzae
that induce cell death in rice. Among 42 tested proteins, five
cell death–inducing proteins were functionally characterized.
The integrative approach described here provides an efficient
strategy for functional identification of fungal cell death–induc-
ing proteins that are involved in plant and fungal interactions.
RESULTS
Gene expression profiling in blast-infected rice leaves.
To obtain more comprehensive gene expression profiles of M.
oryzae during compatible and incompatible interactions with
rice, we constructed one RL-SAGE, eleven MPSS, and seven
SBS libraries for deep sequencing. The RL-SAGE library was
generated from rice (cv. Nipponbare) leaves inoculated with the
compatible isolate Che86061 at 96 h postinoculation (hpi) (Fig.
1A). A total of 18,154 significant signatures were obtained from
the library. Among them, 3,105 (17.1%) and 12,263 (67.5%)
significant signatures matched to the M. oryzae and rice
genomes, respectively. The unmatched signatures may be due to
sequencing errors or are located in the sequencing gaps in both
the genomes. Only 14 signatures matched to both the M. oryzae
and rice genomes, signifying that the majority of signatures
were genome specific. We identified 3,091 signatures specifi-
cally matching to the M. oryzae genome, which correspond to
3,000 previously annotated M. oryzae genes.
Eleven MPSS libraries were generated from the leaves of
wild-type Nipponbare plants or transgenic Nipponbare plants
carrying the blast resistance gene Pi9 (Qu et al. 2006), which
were inoculated with M. oryzae isolate KJ201 during the com-
patible (3, 6, 12, 24, 48, and 96 hpi) or incompatible interac-
tions (3, 6, 12, 24, and 48 hpi) (Fig. 1A). As expected, more
signatures matched to the M. oryzae genome in the compatible
interactions compared with that in the incompatible interac-
tions. A total of 57,671 significant signatures were obtained
from the five incompatible interaction MPSS libraries. Among
these, 724 (1.2%) and 38,024 (65.9%) significant signatures
uniquely matched to the M. oryzae and rice genomes, respec-
tively. From the six compatible interaction libraries, a total of
63,132 significant signatures were obtained. Among them,
2,545 (4%) and 41,784 (66.1%) significant signatures uniquely
matched to the M. oryzae and rice genomes, respectively. Alto-
gether, 3,216 annotated M. oryzae genes were identified from
both the compatible and incompatible MPSS libraries.
The same leaf tissue used for the generation of the MPSS li-
braries (the compatible interaction at 6, 12, 24, and 96 hpi and
the incompatible interaction at 6, 12, and 24 hpi) were used for
the construction of the seven SBS libraries (Fig. 1A). A total
of 65,299 significant signatures were obtained from the three
incompatible-interaction SBS libraries. Among them, 3,492
(5.3%) and 49,706 (76.1%) significant signatures specifically
matched to the M. oryzae genome and rice genome, respec-
tively. A total of 68,825 significant signatures were obtained
from the four compatible-interaction SBS libraries. Signatures
matching to the M. oryzae genome numbered 5,283 (7.7%)
and those matching to the rice genome numbered 50,756
(73.7%). The SBS signatures from both compatible and in-
compatible interactions together identified 4,781 annotated M.
oryzae genes. Altogether, a total of 6,413 annotated M. oryzae
genes expressed during infection process were identified using
the three expression profiling technologies (Fig. 1A).
Identification of genes encoding
putative secreted proteins expressed during infection.
Secreted proteins are known to play essential roles during
fungal-plant interactions (Rep 2005; Zhang and Zhou 2010).
Thus, we focused on the analysis of the secreted protein genes
that were identified in the transcriptome libraries described
above. To obtain most putative secreted protein genes from the
in planta–expressed gene collections, we used two M. oryzae
secreted protein datasets, one by Dean and associates (2005)
(referred to as dataset I) and the other by Choi and associates
(2010) (referred to as dataset II) as references for manual
annotation. A total of 851 distinct secreted protein genes were
identified from both datasets (Fig. 1A, Supplementary Table
S1). About 85.7% (264/308) of the putative secreted protein
genes identified in dataset I were present in dataset II (Fig.
1E). Among the three RL-SAGE, MPSS, and SBS libraries
made from the rice leaves inoculated with compatible isolates
at the same timepoint (96 hpi) (RL-SAGE-96 h, MPSS-96 h,
and SBS-96 h), over two times more secreted protein genes
were identified from the SBS-96 h library than from the other
two libraries in both datasets I and II (Fig. 1C and D).
To gain more insight on the function of in planta–expressed
secreted proteins involved in the rice-M. oryzae interaction, we
conducted a gene ontology (GO)-based classification. This
analysis revealed that most in planta–expressed secreted pro-
teins are associated with metabolic process, followed by devel-
opmental process, cellular process, and multicellular organismal
process (Fig. 2A). For molecular functions, most in planta–
expressed secreted proteins are associated with catalytic activity
and binding (Fig. 2B).
Transient expression assays identified five M. oryzae
apoplastic effectors that induce cell death in rice cells.
Host cell death is a ubiquitous feature in plant-pathogen
interactions. To identify M. oryzae secreted proteins involved
in host cell death, we performed transient expression of M.
oryzae secreted proteins in rice protoplasts, using our estab-
lished method (Chen et al. 2006). In the assay, cell death is
monitored by the reduced expression level of a cotransfected
β-glucuronidase (GUS) reporter gene in rice protoplasts (Fig.
3A) (Dou et al. 2008b; Jia et al. 2000; Mindrinos et al. 1994;
Yoshida et al. 2009). A total of 42 secreted protein genes were
cloned for the transient expression assay (Supplementary Table
S2). For each of the selected genes, two versions of transfec-
tion plasmids were constructed, one containing the full-length

Vol. 26, No. 2, 2013 / 193
open reading frame (ORF) (referred to as FL) and the other
containing the truncated coding region without the signal pep-
tide sequence but with an engineered ATG start codon (referred
to as NS). The transient assay revealed that five out of 42 se-
creted protein genes of the FL version caused a significant
reduction in GUS activity when expressed in rice protoplasts
(Fig. 3B), suggesting that these five proteins can induce cell
death in rice cells. On the other hand, transient expression of
all 42 examined genes of the NS version did not result in any
reduction in cell viability. We thus referred to the five secreted
proteins, MGG_03356, MGG_05531, MGG_07986, MGG_
08409, and, MGG_10234, as MoCDIP1 to MoCDIP5 (M. ory-
zae cell death–inducing proteins), respectively.
We further performed Agrobacterium-mediated transforma-
tion of rice calli with binary vectors containing the five MoCDIP
genes to see whether it was possible to obtain stable transgenic
lines with the expression of these genes for functional analysis.
About 600 calli were used for the transformation of each con-
struct. As expected, no resistant transgenic calli were obtained
after transforming with FL-MoCDIP1, FL-MoCDIP2, FL-
MoCDIP3, or FL-MoCDIP4, individually (data not shown).
By contrast, around 50 to 80 transgenic calli were obtained
from the transformations with NS-MoCDIP1, NS-MoCDIP2,
NS-MoCDIP3, or NS-MoCDIP4, with efficiencies similar to
normal transformation efficiencies (around 8 to 15% for other
constructs) in our lab. Interestingly, transformation with FL-
MoCDIP5 at first yielded a normal efficiency of about 40
transgenic calli with no cell-death phenotypes. However, when
they were cultured on the selection medium for 2 weeks or
longer, the transgenic calli expressing FL-MoCDIP5 started to
show cell-death phenotypes (Fig. 3C). Similar to other NS-
MoCDIP, no cell death was observed in the transgenic calli
Fig. 1. Gene expression profiling of Magnaporthe oryzae during its interaction with rice. A, Summary statistics for gene expression profiling by robust-long
serial analysis of gene expression (RL-SAGE), massively parallel signature sequencing (MPSS), and sequencing by synthesis (SBS), and identification of in
planta–expressed M. oryzae genes encoding putative secreted proteins. B, Clustering analysis of all M. oryzae genes identified in the libraries of RL-SAGE-
96 h, MPSS-96 h, and SBS-96 h, respectively. C, Clustering analysis of putative M. oryzae secreted protein genes retrieved from dataset I (Dean et al. 2005)
in RL-SAGE-96 h, MPSS-96 h, and SBS-96 h, respectively. D, Clustering analysis of putative M. oryzae secreted protein genes retrieved from dataset II
(Choi et al. 2010) in RL-SAGE-96 h, MPSS-96 h, and SBS-96 h, respectively. E, Clustering analysis of putative M. oryzae secreted protein genes in all
timepoints libraries retrieved from datasets I and II, respectively.

194 / Molecular Plant-Microbe Interactions
expressing NS-MoCDIP5 (Fig. 3C). These results, consistent
with the transient expression assay in rice protoplasts, con-
firmed that the five MoCDIP induced cell death when the full-
length proteins are expressed in rice cells.
To functionally investigate the predicted secretion feature of
the five identified MoCDIP, a yeast secretion assay was per-
formed, following the method previously published (Lee et al.
2006). The sequences of both FL-MoCDIP and NS-MoCDIP
Fig. 3. Identification of five in planta–expressed putative secreted proteins that induce cell death in rice cells. A, Schematic representation of the rice proto-
plast transient expression assay approach to the identification of Magnaporthe oryzae secreted proteins that can induce rice cell death. Rice protoplasts were
cotransfected with a reporter β-glucuronidase (GUS) construct (Promoter-GUS-Tnos) and the other construct (Promoter-M.o gene-Tnos) carrying a M. ory-
zae secreted protein gene. Rice cell viability was detected based on monitoring the reduced GUS expression level. MoCDIP = M. oryzae cell death inducing
protein. B, Ectopic expression of the full length of five MoCDIP in rice protoplasts resulted in reduction in cell viability. CK = protoplast sample cotrans-
fected with a GUS reporter and an empty vector control; 1 to 5 = protoplast samples cotransfected with a GUS reporter and the other construct carrying
MoCDIP1 to MoCDIP5, respectively. Data bars show averages from three triplicate samples in one experiment. Each experiment was repeated at least three
times with similar results. C, Ectopic expression of a full-length but not truncated non–signal peptide version of MoCDIP5 resulted in cell death in trans-
genic rice calli. CK = rice calli transformed with empty vector; NS-MoCDIP5 =rice calli transformed with construct carrying a truncated non–signal peptide
version of MoCDIP5; FL-MoCDIP5 = rice calli transformed with construct carrying a truncated full-length MoCDIP5. Pictures were taken at about 20 days
when the newly obtained transgenic calli were maintained on selection media. Transformation experiments were repeated two times, and similar results were
observed. D, Reverse transcription-polymerase chain reaction (RT-PCR) analysis of expression of NS-MoCDIP5 or FL-MoCDIP5 in transgenic rice calli.
CK = RT-PCR result from the rice calli transformed with the empty vector control; 1 to 3 = RT-PCR result from three independent transgenic calli lines.
Fig. 2. Gene ontology (GO) annotation of in planta–expressed Magnaporthe oryzae putative secreted proteins. A, Classification based on biological process.
B, Classification based on molecular function.

Vol. 26, No. 2, 2013 / 195
were fused in frame to the N-terminal end of a yeast invertase
gene (suc2) lacking its own signal peptide sequence. The fusion
constructs were transformed into the yeast strain DBYα2445,
an invertase-deficient mutant (Lee et al. 2006), and the trans-
formed yeasts were grown directly on sucrose medium and
were assayed for secretion. As expected, the yeast strain trans-
formed with the constructs containing the NS-MoCDIP-suc2
fusions did not grow on sucrose medium (Supplementary Fig.
S1), due to the lack of secreted invertase to catalyze the de-
composition of sucrose into fructose and glucose as the carbon
source. In contrast, all five constructs containing the fusions of
the FL-MoCDIP-suc2 enabled the yeast mutant strain to grow
on sucrose medium, confirming that the predicted signal pep-
tides of the five MoCDIP are functional to direct the invertase
fusions to the secretory pathway. These results, together with
the results showing that the signal peptide is required for
MoCDIP expressed in rice cells to induce rice cell death, indi-
cate that these proteins most likely function in the plant apo-
plastic space. However, the exact target site of these proteins in
plant cells remains to be elucidated.
MoCDIP genes are expressed
in infected leaves and appressoria.
To experimentally confirm that the MoCDIP genes are ex-
pressed in infected leaves and to determine their expression
pattern in appressoria and mycelia, reverse transcription-poly-
merase chain reaction (RT-PCR) was carried out using the
RNA extracted from Nipponbare rice leaves inoculated with
the compatible blast isolate KJ201 and from M. oryzae appres-
soria and mycelia. Because of the low proportion of the fungal
mass in the infected leaves at early infection stages, the
Mo28S transcript was not detected before 72 hpi. Similar to
the Mo28S transcript, the MoCDIP2 and MoCDIP4 transcripts
were detected from the infected rice leaves only at 72 hpi, and
the transcripts of MoCDIP1, MoCDIP3, and MoCDIP5 were
detected from the infected rice leaves at 96 hpi (Fig. 4). This
result confirmed that all five MoCDIP were expressed during
infection stages. The transcripts of MoCDIP1 and MoCDIP2
were detected in both appressoria and mycelia with relatively
higher expression in the former, and the transcripts of
MoCDIP3, MoCDIP4, and MoCDIP5 were detected only in
appressoria (Fig. 4).
MoCDIP induce cell death in nonhost plant cells.
Many pathogen effectors induce cell death in nonhost plant
cells (Rep 2005). To determine whether the five MoCDIP have
cell death effects in nonhost plant cells, we performed transi-
ent expression assays of the MoCDIP genes in the protoplasts
of three model plants, maize (Zea mays), Arabidopsis thaliana,
and Nicotianna benthamiana. Consistent with the results ob-
served in rice protoplasts, transient expression of the five FL-
MoCDIP but not the NS-MoCDIP induced a significant reduc-
tion in cell viability in maize protoplasts (Fig. 5A). As for tran-
sient assays in protoplasts of dicot plants Arabidopsis and N.
benthamiana, expression of the FL-MoCDIP, except for FL-
MoCDIP2, caused significant cell-viability reduction. Similar
to the result in rice cells, expression of all five NS-MoCDIP
did not reduce cell viability in either Arabidopsis or N. ben-
thamiana protoplasts (Fig. 5B and C).
We further tested the five MoCDIP in N. benthamiana
leaves via an Agrobacterium-mediated transient-expression
approach. A. tumefaciens containing the empty vector pGD
and a pGD recombinant expressing WtsE were used as nega-
tive and positive controls, respectively. WtsE is a bacterial type
III effector that induces cell death in both host and nonhost
plants (Ham et al. 2008). Consistent with results from the pro-
toplast assays, infiltration of N. benthamiana leaves with the
Agrobacterium strains expressing FL-MoCDIP1, FL-
MoCDIP3, FL-MoCDIP4, or FL-MoCDIP5 resulted in cell-
death responses (Fig. 5D). On the contrary, infiltration of the
FL-MoCDIP2 strain, as well as infiltration of the strains carry-
ing the constructs expressing the NS-MoCDIP, did not result in
cell death in the infiltrated area. RT-PCR analysis was per-
formed to examine the transient expression of the MoCDIP
genes, and the results showed that both the FL-MoCDIP and
NS-MoCDIP genes were expressed at similar levels in the in-
filtrated N. benthamiana leaves (Fig. 5E). These results con-
firmed that MoCDIP1, MoCDIP3, MoCDIP4, and MoCDIP5,
but not MoCDIP2 induce cell death in both monocot and dicot
species.
The timing and appearance of the cell death in N. bentham-
iana leaves induced by the FL-MoCDIP strains were not as
strong as that induced by the WtsE strain. The cell death symp-
tom induced by the WtsE strain usually started at 36 to 48 h
after agroinfiltration, and the symptoms induced by the FL-
MoCDIP1, FL-MoCDIP3, and FL-MoCDIP4 strains generally
appeared at 2 to 3 days after agroinfiltration, with a severe cell
death around the infiltrated site. However, the symptoms in-
duced by FL-MoCDIP5 generally were visible at 4 to 6 days
after agroinfiltration, with weak necrotic spots in the infiltrated
area. This result together with the observation of cell death
only in relative long-term cultured rice calli expressing FL-
MoCDIP5 suggests that FL-MoCDIP5 induces weak cell death
in a delayed pattern.
Physiological basis of the MoCDIP-induced cell death.
Previous studies have shown that plant cell death induced by
some microbial toxins or effectors share some conserved mech-
anisms (Asai et al. 2000; Qutob et al. 2006). To further charac-
terize the physiological properties of the cell death induced by
the MoCDIP, we performed inhibition assays in rice proto-
plasts and N. benthamiana leaves. Two cell death–inducing
proteins, i.e., WtsE and Bax, were also included in the assays
as the controls.
Calcium signaling has been shown to play an important role
in the cell death process (Boudsocq et al. 2010; Lecourieux et
al. 2002). To determine whether calcium signaling is required
for the MoCDIP-induced cell death, LaCl3, a calcium channel
inhibitor, was applied in the inhibition assays. Application of
LaCl3 blocked cell death induced by the transient expression of
all MoCDIP, suggesting that the cell-death process mediated
by these proteins is dependent on a calcium signaling pathway.
Fig. 4. In planta expression pattern of the five Magnaporthe oryzae cell
death–inducing protein genes (MoCDIP). Total RNA samples extracted fro
m
infected rice leaves 0, 24, 48, 72, 96, or 120 h after inoculation, from in
vitro–grown M. oryzae appresorium (A) and mycelium (M) were subjecte
d
to reverse transcription-polymerase chain reaction using specific primers.

196 / Molecular Plant-Microbe Interactions
Light intensity has been demonstrated to be an important fac-
tor for cell-death induction triggered by some toxins or effectors
(Asai et al. 2000; Qutob et al. 2006). To test whether MoCDIP-
induced cell death is light-dependent, we transfected MoCDIP
in rice protoplasts in the light or in the dark. There was no dif-
ference of cell viability between the protoplast samples incu-
bated in either condition, indicating that the cell death process
induced by the MoCDIP in rice protoplasts is light-independent
(Table 1). In contrast, transient expression of the MoCDIP did
not induce any cell-death lesions in the leaves of N. bentham-
iana kept in the dark (Table 1), suggesting that the MoCDIP-
induced cell death in N. benthamiana leaves is light dependent.
Studies have shown that some antiapoptotic proteins such as
BCL-2 family proteins and bax inhibitor-1 (BI-1) can suppress
various types of cell death and are functionally conserved in
yeast, plants, and mammals (Watanabe and Lam 2009). Over-
expression of antiapoptotic proteins was shown to inhibit cell
death induced by multiple stimuli, revealing that various types
of cell death may have a common downstream mechanism
(Dickman et al. 2001). We tested Bcl-xl, a member of the
BCL-2 family, to determine whether MoCDIP-induced cell
death can be inhibited by the antiapoptotic protein. Preinfiltra-
tion with A. tumefaciens cells harboring the Bcl-xl expression
vector did not produce cell-death symptoms after infiltration
Fig. 5. Transient expression of the full-length but not truncated non–signal peptide version of Magnaporthe oryzae cell death inducing protein (MoCDIP) induce
d
cell death in nonhost plant cells. A, B, and C, Transient expression assay of the five MoCDIP in protoplasts of maize, Arabidopsis, and Nicotiana benthamiana,
respectively. CK = protoplast sample cotransfected with a β-glucuronidase (GUS) reporter and an empty vector control; 1 to 5 = protoplast samples cotransfecte
d
with a GUS reporter and the other construct carrying MoCDIP1to MoCDIP5, respectively. D, Transient expression assay of the five MoCDIP in N. benthamiana
leaves by using an agroinfiltration approach. Agroinfiltration was performed on the same leaf side by side with Agrobacterium tumefaciens carrying an empty
vector control (pGD), a positive control (WtsE), constructs with full-length MoCDIP (FL-MoCDIP), or constructs with non–signal peptide sequence
M
oCDI
P
(NS-MoCDIP), respectively. E, Reverse transcription-polymerase chain reaction (RT-PCR) analysis of MoCDIP expression in agroinfiltrated N. benthamiana
leaves. Total RNA was extracted from N. benthamiana leaves at 36 h postinoculation. CK = RT-PCR result from tissues infiltrated with the empty vector con-
trol; 1 = RT-PCR result from tissues infiltrated with FL-MoCDIP; 2 = RT-PCR result from tissues infiltrated with NS-MoCDIP.
Tab le 1 . Inhibition assays of Magnaporthe oryzae cell death–inducing proteins in rice protoplasts and Nicotiana benthamiana leavesa
Applications or treatments
Catalase DPI LaCl3b Dark Bcl_xlc
Effector RP NBL RP NBL RP NBL RP NBL RP NBL
MoCDIP1 + + + + – – + – ND –
MoCDIP2 – ND + ND – ND + ND ND ND
MoCDIP3 + + + + – – + – ND –
MoCDIP4 – – + + – – + – ND –
MoCDIP5 – – + + – – + – ND –
WtsE – – + + – – + + ND –
Bax – – + + – – + + ND –
a RP = rice protoplasts, NBL = N. benthamiana leaves, DPI = diphenyleneiodonium sulfate, + = cell death,–= no cell death, and ND = not determined.
b LaCL3 was dissolved in distilled water, and the same volume of water was applied to rice protoplasts or N. benthamiana leaves as control.
c An empty vector pGD was applied as control. No effects of controls on cell-death assays was observed.

Vol. 26, No. 2, 2013 / 197
Fig. 6. Structural analyses of Magnaporthe oryzae cell death inducing proteins (MoCDIP). Schematic views of MoCDIP and their deletion mutants are
shown on the left. The predicted domains or motifs of MoCDIP are represented as color rectangles. The deleted regions are represented as dotted lines.
MoCDIP and their deletion mutants were transiently expressed in Nicotiana benthamiana leaves or rice protoplasts. Assay results from agroinfiltrated N.
benthamiana leaves or transfected rice protoplasts are shown on the right. The signs +, +–, and – indicate obvious cell death, weak cell death, and no cell
death, respectively. Data bars of protoplast results show averages from three triplicate samples in one experiment. Each experiment was repeated at least
three times with similar results.

198 / Molecular Plant-Microbe Interactions
with A. tumefaciens cells carrying MoCDIP, WtsE, or Bax. In
contrast, N. benthamiana leaves preinfiltrated with the culture
containing the empty vector showed obvious cell-death symp-
toms induced by the transient expression of MoCDIP, WtsE, or
Bax. This result indicated that cell death in N. benthamiana
leaves induced by MoCDIP is suppressed by the antiapoptotic
protein (Table 1).
Sequence and structural analysis of MoCDIP.
Sequence analysis revealed that the five identified MoCDIP
are highly diverse in their sequences and have no sequence ho-
mology with known cell death–inducing effectors in different
pathogens, such as Nep1-like proteins (Qutob et al. 2006),
INF1 (Kamoun et al. 1997), ToxA (Ciuffetti et al. 1997), ToxB
(Martinez et al. 2001), or Nip (Mattinen et al. 2004). BLAST
searches against the M. oryzae database and the NCBI non-
redundant database revealed that MoCDIP2 and MoCDIP4
have a relatively large number of homologs in the sequenced
genomes of M. oryzae and of other organisms (Supplementary
Fig. S2). In contrast, MoCDIP1 and MoCDIP5 have homology
to proteins only from other microorganisms (Supplementary
Fig. S2), and MoCDIP3 has no homologs in the M. oryzae ge-
nome or in the sequenced genomes of other organisms. Homol-
ogy searches also revealed that MoCDIP2 belonged to a family
of CFEM-containing proteins that may function as a cell-sur-
face receptor, signal transducers, or as adhesion molecules in
host-fungi interactions (Kulkarni et al. 2003); MoCDIP4 was a
highly conserved homolog to glycosyl hydrolase family 61
proteins (Davies and Henrissat 1995); and MoCDIP1 and
MoCDIP5 shared similarity to ricin B lectin proteins, although
there was no sequence similarity between the two proteins.
To further delineate the properties of the MoCDIP that are
involved in cell-death induction, we searched conserved do-
mains in the proteins and performed functional analysis of de-
letion mutants based on domain prediction. Deletion mutant
constructs of MoCDIP, except for MoCDIP3, because it does
not contain any predicted domain, were tested in both rice pro-
toplasts and N. benthamiana leaves (Fig. 6). Transient expres-
sion assays revealed that a MoCDIP1 mutant of the N-terminal
region, amino acids (aa) 1 to 185, was sufficient for inducing
cell death in plant cells. However, the N-terminal region, aa 1
to 162, which lacks the PbH1 motif in the aa 1 to 185 frag-
ment, lost the ability to induce cell death, suggesting that the
PbH1 motif may play an important role in inducing cell death
in plants. The N-terminal, CFEM domain-containing region of
MoCDIP2 induced rice cell death efficiently, suggesting that
the predicted GPI anchor was not required for cell-death induc-
tion. As for MoCDIP4, a deletion mutant lacking the cellulose
binding domain (CBD) domain was found to induce only a
weak cell death, and the mutant lacking both the linker frag-
ment and the CBD domain failed to induce cell death, sug-
gesting that the C-terminus of the linker fragment and the CBD
domain are functionally important in inducing cell death in
plants. The assays also showed that MoCDIP5 mutants lacking
the C-terminus or lacking the potential zinc-binding site motif
failed to induce cell death in both rice protoplasts and N. ben-
thamiana leaves, suggesting that the full length of MoCDIP5
is required for cell-death induction in plants.
DISCUSSION
Transcriptome profiling in the rice-M. oryzae interaction.
Using different gene-prediction algorithms, about 12% of
the annotated genes (1,546) are predicted to be putative effec-
tor proteins in the M. oryzae genome (Soanes et al. 2008).
Whether these predicted genes are expressed in infected rice
plants is largely unknown. By applying expressed sequence tag
(EST) analysis, RL-SAGE, and SuperSAGE analysis to infected
rice leaves at the early infection stages, the defense transcrip-
tome in response to M. oryzae infection was characterized
(Ebbole et al. 2004; Gowda et al. 2007; Kim et al. 2001;
Matsumura et al. 2003; Rauyaree et al. 2001). However, these
studies only identified a small number of fungal genes, mainly
because the proportion of the fungal RNA in the infected
tissue was relatively low and the sequencing coverage was not
deep enough. Recently, improved approaches for enrichment
of fungal RNA from infected plant tissues have been employed
to profile the interaction transcriptome of M. oryzae and rice.
Kim and associates (2010) applied EST analysis coupled with
subtractive hybridization of a cDNA library from infected
leaves at very late stages of infection. A total of 712 uniEST
were identified from the fungus, representing up to 31% of the
total uniEST. Mosquera and associates (2009) developed a
procedure to produce heavily infected rice sheaths that allowed
isolation of total RNAs with a high proportion of RNA (up to
20%) originating from biotrophic invasive hyphae of M. ory-
zae. By applying microarray analysis, the authors identified
262 fungal genes and 210 rice genes that were induced up to
10 fold during biotrophic invasion.
In this study, we employed three high-throughput technol-
ogies, i.e., RL-SAGE, MPSS, and SBS, to profile the transcrip-
tome of M. oryzae–infected rice tissue. While RL-SAGE was
only used to study gene-expression profiles from rice leaf tissue
infected with a compatible isolate at 96 hpi, both MPSS and
SBS were applied to study gene expression profiles of rice leaf
tissue inoculated with either a compatible or incompatible iso-
late at different postinoculation timepoints. In the rice leaves at
72 hpi, inoculated with both incompatible and compatible iso-
lates, only a very limited number of fungal genes were identi-
fied, as there were no visible symptoms or only a very few
brown lesions appeared on the rice leaves. When inoculated with
a compatible isolate at 96 hpi, rice leaves developed typical sus-
ceptible blast lesions. Consistently, a relatively large number of
fungal transcripts were detected from heavily infected leaves.
We identified 3,091 (17%), 2,327 (12.3%), and 5,099 (11.4%)
significant M. oryzae tags from the libraries of RL-SAGE-96 h,
MPSS-96 h, and SBS-96 h, respectively. By combining results
from three technologies, we identified a total of 6,413 M. oryzae
genes, including 851 genes that are predicted to encode putative
secreted proteins and might be expressed in planta. Our results
together with those previously reported will provide valuable
information for future studies of the molecular mechanisms
underlying the rice–M. oryzae interaction.
Clustering analysis of the three 96-h libraries showed that
the transcripts recovered from the RL-SAGE, MPSS, and SBS
were partially overlapping, similar to previous results of tran-
scriptional profiles of M. oryzae using MPSS, RL-SAGE, and
oligoarray methods (Gowda et al. 2006). This result also sug-
gests that using multiple different approaches can provide
more comprehensive gene expression profiles. When consider-
ing individual technologies, SBS appeared to be the most effi-
cient method for transcript profiling. While similar numbers of
M. oryzae genes were identified from RL-SAGE-96 h and
MPSS-96 h (3,008 and 2,401 unique M. oryzae genes from the
two libraries, respectively), a total of 4,730 M. oryzae genes
were identified from SBS-96 h (Fig. 1B), almost twice the
gene numbers identified by the other two methods. Moreover,
the M. oryzae transcripts recovered from SBS-96 h included
most of the M. oryzae transcripts from RL-SAGE-96 h or
MPSS-96 h. Our results indicated that many weakly expressed
transcripts of both rice and M. oryzae were recovered in the
libraries, due to the deep coverage of the transcriptome. As the
sequencing cost is rapidly declining, the SBS-based ultra-fast
sequencing method or other next-generation sequencing plat-

Vol. 26, No. 2, 2013 / 199
forms will allow for more in-depth characterization of plant-
pathogen interaction transcriptomes.
Functional identification of M. oryzae effectors
using the rice protoplast transient expression system.
Transcriptional analysis of the M. oryzae effector genes in
infected rice plants has provided a starting point for functional
analysis of the in planta–expressed genes in the rice–M. oryzae
interaction. Over the past two decades, several M. oryzae
avirulence or pathogenicity effector genes have been isolated
by map-based cloning (Kang et al. 1995; Orbach et al. 2000;
Sweigard et al. 1995), genetic association analysis (Yoshida et
al. 2009), or loss-of-function (Ahn et al. 2004; Jeong et al.
2007; Kim et al. 2005; Talbot et al. 1993) approaches. The first
two procedures are time-consuming, tedious, and expensive.
As for loss-of-function approach, it is often hampered by the
fact that many genes may have overlapping functions. For ex-
ample, many knockout mutants of secreted protein genes have
no identifiable phenotype (Mosquera et al. 2009). Thus, a cost-
efficient and high-efficiency gain-of-function method would be
a valuable alternative approach to the identification of M. ory-
zae effectors.
As for gain-of-function identification, the agroinfiltration
transient assay is a widely used approach for characterizing
function of phytopathogen effectors in many solanaceous plants,
especially in N. benthamiana and N. tabacum (Munkvold and
Martin 2009). However, this agroinfiltration method is not
applicable in monocot plants. We previously reported a proto-
plast transient expression system to perform assays directly in
rice cells (Chen et al. 2009a). Recently, Yoshida and associates
(2009) and Okuyama and associates (2011) detected HR in
rice protoplasts coexpressing R gene and its cognate Avr gene
from M. oryzae. Using the system, they successfully screened
the candidates of M. oryzae Avr-Pia, Avr-Pii, Avr-Pik/km/kp,
and rice blast resistance gene Pia. In this study, we identified
five M. oryzae effectors that induce cell death in rice cells. The
results demonstrate that the rice protoplast expression assay is
an efficient method for large-scale screening of putative effec-
tors that induce cell death or HR reaction.
Role of MoCDIP in the interaction
between M. oryzae and rice.
Unlike many bacterial pathogens that deliver effector pro-
teins inside host cells via a type III secretion system, eukary-
otic plant pathogens, like oomycetes and fungi, seem to secrete
a large number of extracellular proteins via the eukaryotic
(type II) secretory pathway (Panstruga and Dodds 2009).
Some secreted proteins are translocated into host cells and
function in the host cytoplasm to suppress host defenses.
Many others function in the host apoplastic space to facilitate
the parasitic lifestyle of pathogens. The latter include degrada-
tive enzymes, toxins, or inhibitors of plant enzymes. More re-
cently, a broader definition of the term effector was suggested
to include these secreted proteins, as they exert some effect on
plant cells (Hogenhout et al. 2009). Over the past few decades,
several apoplastic effectors with toxin or elicitor activity that
can induce cell death in plants have been identified from eu-
karyotic plant pathogens (Rep 2005). Many of these apoplastic
effectors play a positive role in the virulence of the hemibio-
trophic or necrotrophic plant pathogens (Ottmann et al. 2009;
Qutob et al. 2006). From 42 in planta–expressed M. oryzae pu-
tative secreted proteins, we successfully identified five novel
effectors MoCDIP1 to MoCDIP5 that induce plant cell death.
Given the fact that these genes are expressed during infection
stages, especially 96 hpi (Fig. 4), we speculate that some of
these cell death-inducing effectors may facilitate the coloniza-
tion of M. oryzae during the late necrotrophic phase of the
blast infection, which is a common mechanism among differ-
ent pathosystems (Gijzen and Nurnberger 2006). It will be inter-
esting to identify the receptor of these effectors in rice cells
and define the interactions that trigger rice cell necrosis.
The five MoCDIP are highly diverse in their sequences.
While MoCDIP3 does not share any significant similarity with
any known proteins, MoCDIP1, MoCDIP2, MoCDIP4, and
MoCDIP5 have closely related homologs from M. oryzae or
other microorganisms. Our analysis also revealed that cell
death induced by the five different MoCDIP share similar physi-
ological phenotypes, such as the response to light, to inhibitors
of calcium channel, and to Bcl-x1–mediated cell death suppres-
sion. These results together suggest that the cell death–inducing
mechanism of the five MoCDIP might be similar, though their
sequences are quite different. Among the five MoCDIP,
MoCDIP4 belongs to a large family of cellulolytic enzymes
from a wide variety of microorganisms. Related homologous
proteins include endoglucanase, xylanase, and acetylxylan
esterase. Several previous studies have observed that the ex-
pression of genes encoding members of this family of enzymes
was modulated by mitogen-activated protein kinase signaling
pathways, which play critical roles in regulating pathogenesis
as well as other features (Lev and Horwitz 2003; Madhani et
al. 1999; Roberts et al. 2000). However, because of redun-
dancy among cellulolytic enzymes, knock-out of one or even
several genes encoding cellulolytic enzymes had little or no
consequences for virulence (Lev and Horwitz 2003). In this
report, we demonstrate that MoCDIP4, a putative endoglu-
canase, induces cell death when it is expressed in plant cells.
Hence, our finding provides a new clue to the mechanism by
which the cellulolytic enzymes can aid invasion of microor-
ganisms. MoCDIP4 contains two conserved domains, a glycosyl
hydrolase family 61 domain and a fungal CBD. Recently, the
fungal CBD have been shown to be important for the function
of a Phytophthora elicitor lectin CBEL in plant cell-death in-
duction (Gaulin et al. 2006) and for the function of a Tricho-
derma swollenin in plant root colonization (Brotman et al.
2008). More interestingly, the CBD domain was identified as a
novel type of PAMP in microorganisms (Brotman et al. 2008;
Gaulin et al. 2006). Consistent with previous reports, the CBD
domain of MoCDIP4 was found to be important in inducing
plant cell death (Fig. 6), strengthening the notion that the CBD
plays a functionally conserved role, such as serving as a PAMP
elicitor among different plant-pathosystems.
MATERIALS AND METHODS
Plant materials and fungal strains.
Rice (Oryza sativa) materials used in this study were wild-
type Nipponbare plants and transgenic Nipponbare plants car-
rying a blast resistance gene Pi9 (Qu et al. 2006). M. oryzae
isolates used in this study include Che86061 and KJ201.
Construction and data analyses
of RL-SAGE, MPSS, and SBS libraries.
RNA samples isolated from rice leaves inoculated with M.
oryzae isolates were used for library construction. The RL-
SAGE library was constructed following previously described
procedures (Gowda et al. 2004). MPSS library construction
was carried out at Illumina (San Diego, CA, U.S.A.), as de-
scribed (Brenner et al. 2000; German et al. 2008; Meyers et al.
2004). The same total RNAs used for the MPSS library con-
struction (except samples from leaves collected at 3 and 48
hpi) were used for the SBS library construction using our pub-
lished protocols (Venu et al. 2011a).
RL-SAGE clones were sequenced at the Arizona Genome
Institute (Tucson, AZ, U.S.A.). RL-SAGE signatures were

200 / Molecular Plant-Microbe Interactions
identified using the SAGEspy program. MPSS tag processing
was carried out at Illumina (Brenner et al. 2000; Meyers et al.
2004; Lu et al. 2005). The distinct RL-SAGE, MPSS, and SBS
tag sequences were matched to the M. oryzae reference se-
quences, including the whole genomic sequences, annotated
genes, and 500 bp upstream (putative 5′ untranslated region
[UTR]) and downstream (putative 3′ UTR) regions that are
available from the Broad Institute (version 6.0) (Gowda et al.
2006; Venu et al. 2007; Venu et al. 2010, 2011a and b). Rice
and M. oryzae transcript signatures from RL-SAGE, MPSS,
and SBS experiments were separated after matching signatures
to the whole genomic sequence of both rice (Michigan State
University’s Plant Biology directory) and M. oryzea. To iden-
tify in planta–expressed M. oryzae genes encoding for putative
secreted proteins, the experimental RL-SAGE, MPSS, and
SBS signatures were matched to two computational prediction
datasets of M. oryzae secreted proteins (Choi et al. 2010; Dean
et al. 2005). Clustering analysis using Microsoft access was
performed to identify the putative secreted protein genes ex-
pressed specifically or commonly among the three platforms.
The RL-SAGE data were deposited at the Magnaporthe oryzae
community annotation database and the MPSS and SBS data
were deposited at Arabidopsis MPSS Plus database.
Cloning of M. oryzae genes encoding putative secreted
protein and construction of MoCDIP-related vectors.
We first selected about 100 putative secreted protein genes
with high expression levels from the libraries. We had diffi-
culty cloning some of the genes, mainly because the propor-
tion of fungal RNA in the total RNA from infected rice leaves
was relatively low. Some of the genes were cloned from M.
oryzae EST clones or from M. oryzae genomic DNA, for those
with no intron. A total of 42 in planta–expressed M. oryzae pu-
tative secreted protein genes were selected for functional char-
acterization based on the profiling data. The genes were ampli-
fied by PCR, using specific primers, and were cloned into
plant expression vector pXUN (Chen et al. 2009b). MoCDIP-
related vectors were generated based on fragments cloned into
pXUN.
Protoplast transient expression assays.
Transient expression assays in the protoplasts of rice, maize,
Arabidopsis, and N. benthamiana were carried out following
previously described procedures (Chen et al. 2006; Sheen
2001). For transient assays in rice and maize cells, protoplasts
were cotransfected with a maize ubiquitin1 promoter-gus con-
struct (Chen et al. 2006) and the pXUN-based construct of M.
oryzae secreted protein genes, and for transient assays in Arabi-
dopsis and N. benthamiana cells, protoplasts were cotrans-
fected with a Cauliflower mosaic virus 35S promoter-gus con-
struct (Odell et al. 1985) and the pGD-based construct (Goodin
et al. 2002) of M. oryzae secreted protein genes. After trans-
fection, protoplasts were incubated at room temperature for 16
to 24 h. GUS activity was detected essentially as described
(Jefferson et al. 1987), using 4-methylumbelliferyl-β-D-glucu-
ronide (Sigma-Aldrich, St Louis) as the substrate.
More details of materials and methods are available in the
Supplementary Materials and Methods published online.
ACKNOWLEDGMENTS
We are grateful to M. M. Goodin, University of Kentucky, for kindly
providing the pGDG and pGDR plasmids and B. Jaffee for editing the
manuscript. This project is supported by the National Science Foundation-
Plant Genome Research Program (numbers 0605017 and 0701745), and
by the National Natural Science Foundation of China (number 31171808).
S. Chen, P. Songkumarn, and R. C. Venu were involved in the conception
and planning of the study, carried out the experiments, and drafted the
manuscript. M. Gowda, M. Bellizzi, J. Hu, and W. Liu were involved in
performing part of experiments; D. Ebbole, B. C. Meyers, and T. Mitchell
were involved in interpretation of results and writing the manuscript. G.-L.
Wang was responsible for conception and planning of the study, interpreta-
tion of results, and writing the manuscript. All authors read and approved
the final manuscript. The authors declare that they have no competing
interests.
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AUTHOR-RECOMMENDED INTERNET RESOURCES
Arabidopsis MPSS Plus database: mpss.udel.edu/#rice
Broad Institute (version 6.0) download sequence webpage:
www.broadinstitute.org/annotation/genome/magnaporthe_grisea/Multi
Downloads.html
Illumina website: www.illumina.com
Magnaporthe oryzae community annotation database: www.mgosdb.org
Michigan State University’s Plant Biology directory:
ftp.plantbiology.msu.edu/pub/data/Eukaryotic_Projects/o_sativa/annotat
ion_dbs/pseudomolecules/version_6.0
SAGEspy program:
www.osc.edu/research/bioinformatics/projects/sagespy/index.shtml