Plant resistance proteins of the class of nucleotide-binding and leucine-rich repeat domain proteins (NB-LRRs) are immune sensors which recognize pathogen-derived molecules termed avirulence (AVR) proteins. We show that RGA4 and RGA5, two NB-LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae and accomplish different functions in AVR recognition. RGA4 triggers an AVR-independent cell death that is repressed in the presence of RGA5 in both rice protoplasts andNicotiana benthamiana. Upon recognition of the pathogen effector AVR-Pia by direct binding to RGA5, repression is relieved and cell death occurs. RGA4 and RGA5 form homo- and hetero-complexes and interact through their coiled-coil domains. Localization studies in rice protoplast suggest that RGA4 and RGA5 localize to the cytosol. Upon recognition of AVR-Pia, neither RGA4 nor RGA5 is re-localized to the nucleus. These results establish a model for the interaction of hetero-pairs of NB-LRRs in plants: RGA4 mediates cell death activation, while RGA5 acts as a repressor of RGA4 and as an AVR receptor.
Plant activators such as benzothiadiazole (BTH) protect plants against diseases by priming the salicylic acid (SA) signaling pathway. In rice, the transcription factor WRKY45 plays a central role in this process. To investigate the mechanism involved in defense-priming by BTH and the role of WRKY45 in this process, we analyzed the transcripts of biosynthetic genes for diterpenoid phytoalexins (DPs) during the rice–Magnaporthe oryzae interaction. The changes in gene transcription resulted in accumulation of DPs in WRKY45-ox and BTH-pretreated rice after M. oryzae infection. Previously, we reported that cytokinins (CKs), especially isopentenyladenines, accumulated in M. oryzae-infected rice. Here, we show that DP biosynthetic genes are regulated by the SA/CK synergism in a WRKY45-dependent manner. Together, we propose that CK plays a role in mediating the signal of M. oryzae infection to trigger the induction of DP biosynthetic genes in BTH-primed plants.
RAPD analysis was used to characterize the blast fungus populations attacking rice and its associated weeds. Twenty eight M grisea isolates infecting four different hosts Oryza sativa, Eleusine coracana, Echinocloa colonum and Digitaria sanguinalis were collected from contiguous rice fields. DNA of each isolate was amplified using seven random primers. The DNA fingerprints generated using 7 primers suggested that all the isolates were genetically distinct. Cluster analysis delineated the isolates into four distinct groups each corresponding to the host specificity of the isolates. Several host specific bands were also observed. These results indicate that isolates were strictly delimited by their host specificity and gene flow among them was restricted.
In this study, we report the purification, identification, characterization, and gene cloning of a novel hypersensitive response-inducing protein elicitor (MoHrip2) secreted from Magnaporthe oryzae. This 459-bp gene encodes a 152-residue polypeptide that contains an 18-residue signal peptide and exhibits a pI of 4.72 and an apparent molecular mass of 16 kDa. Rice seedlings treated with MoHrip2 exhibited pronounced resistance to M. oryzae compared with control seedlings.
In order to find a natural alternative to the synthetic fungicides currently used against the devastating rice blast fungus, Magnaporthe grisea, this study explored the antifungal potential of citral and its mechanism of action. It was found that citral not only inhibited hyphal growth of M. grisea, but also caused a series of marked hyphal morphological and structural alterations. Furthermore, citral reduced spore germination and germ tube length in a concentration-dependent manner. Following exposure to citral, the hyphal cell surface became wrinkled with folds and cell breakage that were observed under scanning electron microscopy (SEM). There was damage to hyphal cell walls and membrane structures, loss of villous-like material outside of the cell wall, thinning of the cell wall, and discontinuities formed in the cell membrane following treatment based on transmission electron microscopy (TEM).
The fungus Magnaporthe oryzae is the causal agent of a wide range of cereal diseases. For long-term preservation, the fungus is grown and stored desiccated on filter papers at –20 °C. Inoculated filter papers are cut into pieces of 0.5–1.0 cm diameter prior to storage. In the present study, a fast (11 min) and simple method of preparing DNA suitable for amplifying avirulence genes of M. oryzae by polymerase chain reaction (PCR) was developed. A piece of filter paper containing the fungus was removed from a glass bottle and placed in a 0.2 mL Eppendorf tube containing 100 μL 10 × TE. The suspension was heated for 10 min at 95 °C in a PCR machine. The tube was then centrifuged for 1 min at 3000 r min–1. One μL of 10 × TE solution containing DNA was used for PCR. A total of 28 samples were PCR tested. As a positive control, fungal DNA was extracted using a conventional DNA preparation method. DNA samples obtained from both methods were stored at 4 °C. PCR was performed with DNA on the preparation day and after 4, 8, 10, and 18 days of refrigerated storage. In four samples, samples 12, 13, 14, and 28, AVR-Pi9 failed to be amplified. These four samples were tested with a different set of primers for AVR-Pi9, and for AVR-Pita1, confirming that the quality of the samples was insufficient for PCR. Overall, for nearly 90% (24/28) of the samples, the quality of the DNA prepared directly from the fungus on filter paper appeared suitable for a rapid survey of genetic identity of the rice blast fungus by PCR. This method will be useful and effective for reducing cost and time and could readily be adopted worldwide for analysis of M. oryzae and possibly other fungi.
Diverse pesticidal activities of anthothecol derived from Khaya anthotheca (Meliaceae) and three limonoids (gedunin, limonin and obacunone) were determined using six phytopathogenic fungi (Pyricularia grisea, Rhizoctonia solani, Botrytis cinerea,. Puccinia recondita, Phytophthora, ... ) and four insect pests (Plutella xylostella).
Associated diazotrophic bacteria have been shown to contribute with fixed atmospheric nitrogen to the rice crop making possible to reduce the application of nitrogen fertilizers, limiting with it the growth of Pyricularia oryzae. In addition, the metabolites released by these bacteria in the rhizosphere make the plague concentration and the disease severity decrease leading to the reduction of chemical product applications and a lower incidence of rice blast under field conditions. Cuban autochthonous strains have the ability to control Pyricularia oryzae in vitro, which confirms the fact that associated diazotrophic bacteria stimulate plant growth and mitigate the deleterious effects caused by this disease in rice.
TYROSINE DEPHOSPHORYLATION OF OSMPK6 IS A NODE OF ABSCISIC-ACID-MEDIATED SUPPRESSION OF SALICYLIC-ACID DEFENSE SIGNALING IN RICE
Phosphorylation/activation of a rice mitogen-activated protein kinase (MAPK), OsMPK6, by dexamethasone-induced expression of constitutively active OsMKK10-2 mimicked the activation of the SA pathway in rice, leading to blast resistance dependent on WRKY45, the central transcription factor in the rice SA pathway. Conversely, ABA treatment dephosphorylated OsMPK6 at its tyrosine residue in rice seedlings. Two protein tyrosine phosphatases, OsPTP1 and -2 were found to dephosphorylate and inactivate OsMPK6 in vitro. In wild-type rice, low temperature, as well as ABA, suppressed the blast resistance induced by a chemical defense inducer, benzothiadiazole. However, simultaneous knockdown of OsPTP1 and -2 by RNA interference abolished the suppression of benzothiadiazole-induced blast resistance by both ABA and low temperature. These results not only show that tyrosine dephosphorylation of OsMPK6 by PTPases is a node of the antagonistic interaction of cold-induced ABA signaling against SA defense signaling in rice, but also provides an effective way to prevent the damages by blast disease under low temperature.
RECOGNITION OF THE MAGNAPORTHE ORYZAE EFFECTOR AVRPIZ-T BY MULTIPLE HOST TARGETS IN RICE
In the last few years, we have applied an integrated approach to dissecting the AvrPiz-t and Piz-t interaction and identified several AvrPiz-t-interacting proteins (APIPs). Among them, APIP6, a RING finger E3 ligase, degrades AvrPiz-t and is a positive regulator of PAMP-triggered immunity (PTI). APIP5 encodes a transcription factor and negatively regulates cell death and disease resistance to M. oryzae.
CHEMICAL COMMUNICATION INVOLVED IN MAGNAPORTHE-RICE INTERACTION DURING INITIATION OF BLAST DISEASE
Recently, we identified a “pathogenicity island” in the vicinity of the ABC3 locus that harbors a Polyketide Synthase and a Cytochrome P450-related locus essential for Magnaporthe pathogenesis. Loss of PKS1 leads to hypermelanized appressoria and defects in septin localization and penetration pore formation. Pks1-GFP is cytosolic and present exclusively in the appressoria. Inhibition of melanin synthesis affects Pks1-GFP expression and localization. Thus, it is likely that a substrate of Pks1 is essential for host penetration and is shared with the melanin biosynthesis pathway.
INFECTION ASSOCIATED AUTOPHAGY IS INDEPENDENT OF APOPTOSIS AND SUFFICIENT FOR APPRESSORIUM-MEDIATED PLANT INFECTION BY MAGNAPORTHE ORYZAE
Here, we show that ultrastructural and biochemical changes characteristic of apoptosis occur in a M. oryzae in response to pro-apototic stimuli. We identified a number of apoptosis associated genes in M.oryzae and targeted these gene for deletion to determine whether their lack of function has any physiological consequences for the fungus in terms of its response to stress, and in its capacity to infect. M.oryzae has two metacaspase genes MCP1 and MCP2. We found that the single deletion mutants mcp1 and mcp2 and the double mutant mcp1/mcp2 were not affected in their sensitivity to a range of pro-apoptotic stimuli but did show reduced growth in the presence of agents that disrupt endoplasmic reticulum homeostasis, and in addition were fully pathogenic. We have also deleted a number of genes associated with caspase independent apoptosis. These mutants all resulted in an altered response to various apoptotic stresses but retained wild-type virulence. We conclude that although apoptosis occurs in M.oryzae the cell death of the conidium which is a pre-requisite for infection is accomplished solely by autophagy.
MAGNAPORTHE - RICE INTERACTIONS AS REVEALED BY WHOLE GENOME ANALYSIS
Using whole genome information of M. oryzae and rice, we isolated AVR-Pia, AVR-Pii, AVR-Pik and cognate R-genes Pia and Pii. In this talk, I introduce whole genome sequence (WGS)-based gene isolation methodologies including MutMap and report our latest findings on the interaction between AVR-Pik and Pik.
Deletion of MoFKH1 (ΔMofkh1) resulted in reduced mycelial growth and conidial germination, abnormal septation and stress response, and reduced virulence. Similarly, ΔMohcm1 exhibited reduced mycelial growth and conidial germination. On the other hand, loss of MoFOX1 (ΔMofox1) did not show any noticeable changes in development, pathogenicity, and stress response. Deletion of MoFOX2 was not successful even after repeated attempts. Taken together, these results suggested that MoFKH1 and Mo- HCM1 are important in fungal development and that MoFKH1 is further implicated in pathogenicity and stress response in M. oryzae.
Transgenic over-expression of HvSNAC1 in barley cv. Golden Promise reduced the severity of Ramularia leaf spot (RLS), caused by the fungus Ramularia collo-cygni, but had no effect on disease symptoms caused by Fusarium culmorum, Oculimacula yallundae (eyespot), Blumeria graminis f. sp. hordei (powdery mildew), or Magnaporthe oryzae (blast)
Here, we report a novel regulatory mechanism of MAPK phosphorylation and activation besides the canonical MAPK cascade. A rice (Oryza sativa) calcium-dependent protein kinase (CDPK), CPK18, was identified as an upstream kinase of MAPK (MPK5) in vitro and in vivo. Curiously, CPK18 was shown to phosphorylate and activate MPK5 without affecting the phosphorylation of its TXY motif. Instead, CPK18 was found to predominantly phosphorylate two Thr residues (Thr-14 and Thr-32) that are widely conserved in MAPKs from land plants. Further analyses reveal that the newly identified CPK18-MPK5 pathway represses defense gene expression and negatively regulates rice blast resistance. Our results suggest that land plants have evolved an MKK-independent phosphorylation pathway that directly connects calcium signaling to the MAPK machinery.
The fungus Harpophora oryzae is a close relative of the pathogen Magnaporthe oryzae and a beneficial endosymbiont of wild rice. Here, we show that H. oryzae evolved from a pathogenic ancestor. The overall genomic structures of H. and M. oryzae were found to be similar. However, during interactions with rice, the expression of 11.7% of all genes showed opposing trends in the two fungi, suggesting differences in gene regulation. Moreover, infection patterns, triggering of host defense responses, signal transduction and nutritional preferences exhibited remarkable differentiation between the two fungi. In addition, the H. oryzae genome was found to contain thousands of loci of transposon-like elements, which led to the disruption of 929 genes. Our results indicate that the gain or loss of orphan genes, DNA duplications, gene family expansions and the frequent translocation of transposon-like elements have been important factors in the evolution of this endosymbiont from a pathogenic ancestor.
A small and extra chromosome of 1.6 Mb was previously identified in a Pyricularia oryzae strain, 84R-62B. To understand a role of the 1.6 Mb chromosome in the pathogenic changeability of P. oryzae, we performed experiments designed to characterize the 1.6 Mb chromosome. A gene family encoding secreted protein Pex31s in P. oryzae consists of five homologs,Pex31-A to -E. Among them, Pex31-A and -D are known to be recognized by Pik-m and Pik/Pik-m/Pik-p, respectively. In the present study, we identified Pex31-A and -D in the genome of 84R-62B. The present study is the first report showing that loss of a small and extra chromosome leads to pathogenic mutation of P. oryzae and may provide a new insight into the mechanisms generating pathogenic variation of this fungus.
We identified a transcription factor in rice, OsNAC111, belonging to the TERN subgroup of the NAC family that was transcriptionally upregulated after rice blast fungus inoculation. Transgenic rice plants overexpressing OsNAC111 showed increased resistance to the rice blast fungus. In OsNAC111-overexpressing plants, the expression of several defense-related genes including pathogenesis-related (PR) genes was constitutively high compared with the control. These genes all showed blast disease-responsive expression in leaves. These results indicate that OsNAC111 positively regulates the expression of a specific set of PR genes in the disease response and contributes to disease resistance.
Approximately 100 000 putative mutants of rice (Oryza sativa L.) have been generated by mutagens. Mutant genes involved in plant architecture, grain quality and disease resistance have been isolated and characterized. In this review, we described the ethyl methanesulfonate (EMS), irradiation, and fast neutron methods used to create rice mutants; methods for the analysis of rice genes that are responsible for mutations; the use of new mutants for rice breeding and functional genomics; and the molecular mechanisms of blast resistance gene-mediated defence responses.
We performed a comparative genomic analysis that identified 74 NADK gene homologs from 24 species representing the eight major plant lineages within the supergroup Plantae: glaucophytes, rhodophytes, chlorophytes, bryophytes, lycophytes, gymnosperms, monocots and eudicots. OsNADK genes is induced to varying degrees by abiotic stress such as cold, heat, drought (PEG), salt (NaCl) and oxidative (methyl viologen, MV), as well as by biotic stress such as the pathogens Magnaporthe oryzae.