Pathogenicity Of Rice Blast Fungus Research Articles

Multi-omics analysis revealed that the protein kinase MoKin1 affected the cellular response to endoplasmic reticulum stress in the rice blast fungus, Magnaporthe oryzae

BackgroundPrevious studies have shown that protein kinase MoKin1 played an important role in the growth, conidiation, germination and pathogenicity in rice blast fungus, Magnaporthe oryzae. ΔMokin1 mutant showed significant phenotypic defects and significantly reduced pathogenicity. However, the internal mechanism of how MoKin1 affected the development of physiology and biochemistry remained unclear in M. oryzae.ResultThis study adopted a multi-omics approach to comprehensively analyze MoKin1 function, and the results showed that MoKin1 affected the cellular response to endoplasmic reticulum stress (ER stress). Proteomic analysis revealed that the downregulated proteins in ΔMokin1 mutant were enriched mainly in the response to ER stress triggered by the unfolded protein. Loss of MoKin1 prevented the ER stress signal from reaching the nucleus. Therefore, the phosphorylation of various proteins regulating the transcription of ER stress-related genes and mRNA translation was significantly downregulated. The insensitivity to ER stress led to metabolic disorders, resulting in a significant shortage of carbohydrates and a low energy supply, which also resulted in severe phenotypic defects in ΔMokin1 mutant. Analysis of MoKin1-interacting proteins indicated that MoKin1 really took participate in the response to ER stress.ConclusionOur results showed the important role of protein kinase MoKin1 in regulating cellular response to ER stress, providing a new research direction to reveal the mechanism of MoKin1 affecting pathogenic formation, and to provide theoretical support for the new biological target sites searching and bio-pesticides developing.

Csn5 inhibits autophagy by regulating the ubiquitination of Atg6 and Tor to mediate the pathogenicity of Magnaporthe oryzae

Csn5 is subunit 5 of the COP9 signalosome (CSN), but the mechanism by which it strictly controls the pathogenicity of pathogenic fungi through autophagy remains unclear. Here, we found that Csn5 deficiency attenuated pathogenicity and enhanced autophagy in Magnaporthe oryzae. MoCSN5 knockout led to overubiquitination and overdegradation of MoTor (the core protein of the TORC1 complex [target of rapamycin]) thereby promoted autophagy. In addition, we identified MoCsn5 as a new interactor of MoAtg6. Atg6 was found to be ubiquitinated through linkage with lysine 48 (K48) in cells, which is necessary for infection-associated autophagy in pathogenic fungi. K48-ubiquitination of Atg6 enhanced its degradation and thereby inhibited autophagic activity. Our experimental results indicated that MoCsn5 promoted K48-ubiquitination of MoAtg6, which reduced the MoAtg6 protein content and thus inhibited autophagy. Aberrant ubiquitination and autophagy in ΔMocsn5 led to pleiotropic defects in the growth, development, stress resistance, and pathogenicity of M. oryzae. In summary, our study revealed a novel mechanism by which Csn5 regulates autophagy and pathogenicity in rice blast fungus through ubiquitination.

Development of a SCAR marker linked to fungal pathogenicity of rice blast fungus Magnaporthe Oryzae.

PCR-based molecular approaches including RAPD (random amplified polymorphic DNA), ISSR (inter-simple sequence repeat), and SRAP (sequence-related amplified polymorphism) are commonly used to analyze genetic diversity. The aims of this study are to analyze genetic diversity of M. oryzae isolates using PCR-based molecular approaches such as RAPD, ISSR, and SRAP and to develop SCAR marker linked to the pathogenicity of rice blast fungus. Twenty Magnaporthe oryzae isolates were collected mainly from the south of Vietnam and assessed for genetic variation by RAPD, ISSR, and SRAP methods. The comparison of those methods was conducted based on the number of polymorphic bands, percentage of polymorphism, PIC values, and phylogenetic analysis. Then, sequenced characterized amplified region (SCAR) markers were developed based on specific bands linked to fungal pathogenicity of rice blast fungus, M. oryzae. The results indicated that SRAP markers yielded the greatest number of polymorphic bands (174) and occupied 51.7% with polymorphism information content (PIC) value of 0.66. Additionally, the SRAP approach showed stability and high productivity compared with RAPD and ISSR. The SCAR marker developed from the SRAP method identified the presence of the avirulence AVR-pita1 gene involving fungal pathogenicity that can break down blast resistance in rice cultivars. The consistency of SCAR marker obtained in this study showed its efficiency in rapid in-field detection of fungal pathogenicity. SCAR marker developed from SRAP technique provides a useful tool for improving the efficiency of blast disease management in rice fields.

The inhibitor of apoptosis protein MoBir1 is involved in the suppression of hydrogen peroxide-induced fungal cell death, reactive oxygen species generation, and pathogenicity of rice blast fungus

The inhibitor of apoptosis protein (IAP) family has been identified in a variety of organisms. All IAPs contain one to three baculoviral IAP repeat (BIR) domains, which are required for anti-apoptotic activity. Here, we identified a type II BIR domain-containing protein, MoBir1, in the rice blast fungus Magnaporthe oryzae. Expression of the MoBIR1 gene in Saccharomyces cerevisiae suppressed hydrogen peroxide-induced cell death and delayed yeast cell chronological aging. Delayed aging was found to require the carboxyl terminus of MoBir1. M. oryzae transformants overexpressing the MoBIR1 gene demonstrated increased growth rate and biomass, delayed mycelial aging, and enhanced resistance to hydrogen peroxide but reduced reactive oxygen species generation and virulence. Moreover, MoBIR1-overexpressing transformants exhibited anti-apoptotic activity. However, MoBIR1 silencing resulted in no obvious phenotypic changes, compared with the wild-type M. oryzae strain Guy11. Our findings broaden the knowledge on fungal type II BIR domain-containing proteins.

A proteomic insight into the MSP1 and flg22 induced signaling in Oryza sativa leaves

Previously, we reported a novel Magnaporthe oryzae- secreted protein MSP1, which triggers cell death and pathogen-associated molecular pattern (PAMP)-triggered immune (PTI) responses in rice. To investigate the MSP1 induced defense response in rice at the protein level, we employed a label-free quantitative proteomic approach, in parallel with flg22 treatment, which is a well-known elicitor. Exogenous application of MSP1 to rice leaves induced an oxidative burst, MAPK3/6 activation, and activation of pathogenesis-related genes (DUF26, PBZ, and PR-10). MaxQuant based label free proteome analysis led to the identification of 4167 protein groups of which 433 showed significant differences in response to MSP1 and/or flg22 treatment. Functional annotation of the differential proteins showed that majority of the proteins related to primary, secondary, and lipid metabolism were decreased, while proteins associated mainly with the stress response, post-translational modification and signaling were increased in abundance. Moreover, several peroxidases and receptor kinases were induced by both the elicitors, highlighting their involvement in MSP1 and flg22 induced signaling in rice. Taken together, the results reported here contribute to our understanding of MSP1 and flg22 triggered immune responses at the proteome level, thereby increasing our overall understanding of PTI signaling in rice. Biological significanceMSP1 is a M. oryzae secreted protein, which triggers defense responses in rice. Previous reports have shown that MSP1 is required for the pathogenicity of rice blast fungus, however, the exact mechanism of its action and its downstream targets in rice are currently unknown. Identification of the downstream targets is required in order to understand the MSP1 induced signaling in rice. Moreover, key proteins identified could also serve as potential candidates for the generation of disease resistance crops by modulating stress signaling pathways. Therefore, here we employed, for the first time, a label-free quantitative proteomic approach to investigate the MSP1 induced signaling in rice together with flg22. Functional annotation of the differential proteins showed that majority of the proteins related to primary, secondary, and lipid metabolism were decreased, while proteins related to the defense response, signaling and ROS detoxification were majorly increased. Thus, as an elicitor, recombinant MSP1 proteins could be utilized to inducing broad pathogen resistance in crops by priming the local immune responses.

イネいもち病菌の病原力(性)と菌類ウイルスの作用とその応用

イネいもち病菌の病原力(性)と菌類ウイルスの作用とその応用

Characterization of Molecular Identity and Pathogenicity of Rice Blast Fungus in Hunan Province of China

The blast (Magnaporthe oryzae) resistance (R) gene is the most economical and environmental method to control rice blast disease. Characterization of molecular identity and pathogenicity of M. oryzae benefits the deployment of effective blast R genes. In order to identify blast R genes that would be effective in Hunan Province,182 M. oryzae strains were analyzed with a Chinese differential system (CDS), repetitive element-based polymerase chain reaction (rep-PCR), and the presence and absence of avirulence (AVR) genes by PCR amplification with gene-specific primers. Identified blast R genes were validated with 24 monogenic lines (ML) carrying 24 major R genes. In total, 28 races (isolates) of M. oryzae was identified with CDS, and classified into 20 distinct groups with rep-PCR. Interestingly, AVR-Pia, AVR-Pik, AVR-Pizt, AVR-Pib, and AVR-Pi9 were detected in more than 86.8% of the isolates; AVR-Pita1 was in 51.3% and AVR-Pii was in only 2.5%. In contrast, pathogenicity assays on 24 ML demonstrated that Pi9, Piz5, Pikh, and Pikm were more effective, with resistant frequencies of 91.6, 91, 87.9, and 87.3%, respectively; Pia, Piks, Pit, Pi12, and Pib were less than 15%. These findings revealed the complexity of a genetic basis of rice blast resistance, and shed light on useful blast R genes in Hunan Province.

Magnaporthe oryzae-Secreted Protein MSP1 Induces Cell Death and Elicits Defense Responses in Rice.

The Magnaporthe oryzae snodprot1 homolog (MSP1), secreted by M. oryzae, is a cerato-platanin family protein. msp1-knockout mutants have reduced virulence on barley leaves, indicating that MSP1 is required for the pathogenicity of rice blast fungus. To investigate the functional roles of MSP1 and its downstream signaling in rice, recombinant MSP1 was produced in Escherichia coli and was assayed for its functionality. Application of MSP1 triggered cell death and elicited defense responses in rice. MSP1 also induced H2O2 production and autophagic cell death in both suspension-cultured cells and rice leaves. One or more protein kinases triggered cell death, jasmonic acid and abscisic acid enhanced cell death, while salicylic acid suppressed it. We demonstrated that the secretion of MSP1 into the apoplast is a prerequisite for triggering cell death and activating defense-related gene expression. Furthermore, pretreatment of rice with a sublethal MSP1 concentration potentiated resistance to the pathogen. Taken together, our results showed that MSP1 induces a high degree of cell death in plants, which might be essential for its virulence. Moreover, rice can recognize MSP1, resulting in the induction of pathogen-associated molecular pattern-triggered immunity.

Skp1, a component of E3 ubiquitin ligase, is necessary for growth, sporulation, development and pathogenicity in rice blast fungus (Magnaporthe oryzae)

Ubiqitination is an important process in eukaryotic cells involving E3 ubiquitin ligase, which co-ordinates with cell cycle proteins and controls various cell functions. Skp1 (S-phase kinase-associated protein 1) is a core component of the SCF (Skp1-Cullin 1-F-box) E3 ubiquitin ligase complex necessary for protein degradation by the 26S proteasomal pathway. The rice blast fungus Magnaporthe oryzae has a single MoSKP1(MGG_04978) required for viability. Skp1 has multiple functions; however, its roles in growth, sporulation and appressorial development are not understood. MoSKP1 complements Skp1 function in the fission yeast temperature-sensitive mutant skp1 A7, restoring the normal length of yeast cells at restrictive temperature. The MoSkp1 protein in M. oryzae is present in spores and germ tubes, and is abundantly expressed in appressoria. Various RNA interference (RNAi) and antisense transformants of MoSKP1 in B157 show reduced sporulation, defective spore morphology, lesser septation and diffuse nuclei. Further, they show elongated germ tubes and are unable to form appressoria. Transformants arrested in G1/S stage during initial spore germination show a similar phenotype to wild-type spores treated with hydroxyurea (HU). Reduced MoSkp1 transcript and protein levels in knockdown transformants result in atypical germ tube development. MoSkp1 interacts with the putative F-box protein (MGG_06351) revealing the ability to form protein complexes. Our investigation of the role of MoSKP1 suggests that a decrease in MoSkp1 manifests in decreased total protein ubiquitination and, consequently, defective cell cycle and appressorial development. Thus, MoSKP1 plays important roles in growth, sporulation, appressorial development and pathogenicity of M. oryzae.

The syntaxin protein (MoSyn8) mediates intracellular trafficking to regulate conidiogenesis and pathogenicity of rice blast fungus.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate cellular membrane fusion and intracellular vesicle trafficking in eukaryotic cells, and are critical in the growth and development of pathogenic fungi such as Magnaporthe oryzae which causes rice blast. Rice blast is thought to involve distinct SNARE-mediated transport and secretion of fungal effector proteins into the host to modulate rice immunity. We have previously characterized two SNARE proteins, secretory protein (MoSec22) and vesicle-associated membrane protein (MoVam7), as being important in cellular transport and pathogenicity. Here, we show that syntaxin 8 (MoSyn8), a Qc-SNARE protein homolog, also plays important roles in growth, conidiation, and pathogenicity. The MoSYN8 deletion mutant (∆Mosyn8) mutant exhibits defects in endocytosis and F-actin organization, appressorium turgor pressure generation, and host penetration. In addition, the ∆Mosyn8 mutant cannot elaborate biotrophic invasion of the susceptible rice host, or secrete avirulence factors Avr-Pia (corresponding to the rice resistance gene Pia) and Avrpiz-t (the cognate Avr gene for the resistance gene Piz-t) proteins. Our study of MoSyn8 advances our understanding of SNARE proteins in effector secretion which underlies the normal physiology and pathogenicity of M.oryzae, and it sheds new light on the mechanism of the blight disease caused by M.oryzae.

Parasexual Recombination in Magnaporthe oryzae

Parasexual recombination is thought to be one of the causes of variation in the pathogenicity of rice blast fungus, Magnaporthe oryzae, which breaks down resistant rice varieties. In this study, the virulence and fitness of parasexual recombinants of M. oryzae were examined in vitro and in the field. According to the results of the genetic analysis of the pathogenicity of parasexual recombinants of the fungus, the segregation ratios of avirulence and virulence among recombinants on rice cultivar Hattan 3 and line K59-1 were consistent with those of the sexual progeny of the fungus. This indicated that the avirulent genes of the parasexual recombinants were segregated in a manner similar to that in sexual reproduction. The fitness of parasexual recombinants derived from co-cultures of parental isolates was also investigated. The recombinants produced intermediate-sized lesions and intermediate numbers of spores between the parents on the host. Plants inoculated with the recombinant also showed intermediate disease severities between the parents in the field. Parasexual recombinants can attack a rice multiline system because their virulence and fitness is inherited from the parents.

A novel ABC transporter gene ABC2 involved in multidrug susceptibility but not pathogenicity in rice blast fungus, Magnaporthe grisea

We cloned a novel ATP-binding cassette (ABC) transporter gene ABC2 from the rice blast fungus, Magnaporthe grisea. ABC2 protein had nucleotide-binding folds (NBF) and predicted transmembrane domains (TMD 6) arranged in a duplicate [NBF–TMD 6] 2 configuration and showed the highest amino acid homology with BMR1 of Botrytis cinerea. Transcription of the gene was up-regulated by treatment with many toxicants, including several blasticides, demethylation inhibitors (DMI), and antibiotics. ABC2 disruptants displayed an increased sensitivity to bitertanol, myclobutanil, and tebuconazole (DMIs), camptothecin (alkaloid) and cycloheximide (antibiotic). This result demonstrated that ABC2 is a member of multidrug transporters. We performed infection assay of ABC2 disruptants towards rice. However, no obvious difference of disease severities between ABC2 disruptants and wild-type strain was observed, demonstrating that ABC2 is not involved in pathogenicity.

Pathogenicity of rice blast fungus collected from foreign countries

Pathogenicity of rice blast fungus collected from foreign countries