Biochemical defence responses of resistant and susceptible rice genotypes against blast pathogen Magnaporthe oryzae

Abstract

Rice blast is the leading fungal disease which is caused by Magnaporthe oryzae that contributes for the significant decline in the rice yield throughout the globe. There is a need for the understanding of biochemical changes in rice plant during blast infection for the development of novel disease control strategies. In the present study, we isolated M. oryzae from the local paddy fields and the fungal isolates (VCF and PON) were identified by ITS-PCR using genomic DNA samples. Further, we inoculated resistant (BR2655 and TUNGA) and susceptible (INTAN and HR12) rice cultivars with PON and VCF isolates. PON isolate showed relatively high virulence compared to VCF and standard MTCC fungal strains. Therefore, we evaluated the effect of PON on the total protein content and plant defence-related key enzymes (peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, β-glucosidase, chitinase and lipoxygenase) activities between 24- and 120-hour post-inoculation (hpi). The results demonstrated the decrease in total protein content in all the inoculated cultivars. In addition, we observed the variation in the activity of peroxidase, polyphenol oxidase, β-glucosidase, chitinase and lipoxygenase at different time points in all the tested rice plants compared to respective controls. However, no significant difference was observed in the phenylalanine ammonia lyase activity relative to its control. Taken together, this study emphasizes on the variation in the activities of plant defence enzymes in different plant cultivars against the tested fungal pathogen and also implementation of defence enzymes as biochemical markers for resistant breeding.     

稻瘟菌无毒基因研究进展

稻瘟菌是引起水稻稻瘟病的病原物。水稻与稻瘟菌间存在广泛而特异的相互作用,是研究寄主与病原物互作的重要模式系统。本文对稻瘟菌与水稻互作最重要的激发子―无毒基因的研究现状进行了概括,讨论了无毒基因的定位、克隆方法以及已克隆无毒基因的功能及进化研究,同时对今后无毒基因研究的重要方向进行了探讨,为深入理解无毒基因的功能及与水稻可能的互作关系奠定了基础。     

Inhibitory Action of Biofungicide Physcion on Initial and Secondary Infection of Magnaporthe oryzae

Rice blast, caused by the ascomycete fungus Magnaporthe oryzae, is one of the most destructive plant diseases in the world and a serious threat to global food security. Biofungicide physcion, extracted from roots of Chinese traditional herb rhubarb, has been commercialized to control cucumber powdery mildew (Podosphaera fuliginea) in China as a new active and environmentally‐friendly ingredient. This study investigated its bioactivity on rice blast and impact on the infection of M. oryzae. Physcion was effective on M. oryzae with 50% inhibition concentration of 18.19 mg/l on the fungus mycelium growth in vitro and provided 86 and 98% control efficacy in vivo at the concentrations of 40 and 60 mg/l, respectively. It inhibited the initial infection of M. oryzae by inhibiting growth of mycelia, the germination of conidia and the formation of appressoria. Moreover, it inhibited the secondary infection of the fungus at the concentration of 40 mg/l, but had no influence at the low concentrations of 1.25–20 mg/l. So physcion might be a promising natural plant extract that is worthy of being explored to apply on the control of rice blast.     

Role of lysine residues of the Magnaporthe oryzae effector AvrPiz-t in effector- and PAMP-triggered immunity

Magnaporthe oryzae is an important fungal pathogen of both rice and wheat. However, how M. oryzae effectors modulate plant immunity is not fully understood. Previous studies have shown that the M. oryzae effector AvrPiz-t targets the host ubiquitin-proteasome system to manipulate plant defence. In return, two rice ubiquitin E3 ligases, APIP6 and APIP10, ubiquitinate AvrPiz-t for degradation. To determine how lysine residues contribute to the stability and function of AvrPiz-t, we generated double (K1,2R-AvrPiz-t), triple (K1,2,3R-AvrPiz-t) and lysine-free (LF-AvrPiz-t) mutants by mutating lysines into arginines in AvrPiz-t. LF-AvrPiz-t showed the highest protein accumulation when transiently expressed in rice protoplasts. When co-expressed with APIP10 in Nicotiana benthamiana, LF-AvrPiz-t was more stable than AvrPiz-t and was less able to degrade APIP10. The avirulence of LF-AvrPiz-t on Piz-t:HA plants was less than that of AvrPiz-t, which led to resistance reduction and lower accumulation of the Piz-t:HA protein after inoculation with the LF-AvrPiz-t-carrying isolate. Chitin- and flg22-induced production of reactive oxygen species (ROS) was higher in LF-AvrPiz-t than in AvrPiz-t transgenic plants. In addition, LF-AvrPiz-t transgenic plants were less susceptible than AvrPiz-t transgenic plants to a virulent isolate. Furthermore, both AvrPiz-t and LF-AvrPiz-t interacted with OsRac1, but the suppression of OsRac1-mediated ROS generation by LF-AvrPiz-t was significantly lower than that by AvrPiz-t. Together, these results suggest that the lysine residues of AvrPiz-t are required for its avirulence and virulence functions in rice.     

Disruption of actin motor function due to MoMyo5 mutation impairs host penetration and pathogenicity in Magnaporthe oryzae

Actin motor myosin proteins are the driving forces behind the active transport of vesicles, and more than 20 classes of myosin have been found to contribute to a wide range of cellular processes, including endocytosis and exocytosis, autophagy, cytokinesis and the actin cytoskeleton. In Saccharomyces cerevisiae, class V myosin Myo2 (ScMyo2p) is important for the transport of distinct sets of cargo to regions of the cell along the cytoskeleton for polarized growth. To study whether myosins play a role in the formation or function of the appressorium (infectious structure) of the rice blast fungus Magnaporthe oryzae, we identified MoMyo5 as an orthologue of ScMyo2p and characterized its function. Targeted gene disruption revealed that MoMyo5 is required for intracellular transport and is essential for hyphal growth and asexual reproduction. Although the ΔMomyo5 mutant could form appressorium‐like structures, the structures were unable to penetrate host cells and were therefore non‐pathogenic. We further found that MoMyo5 moves dynamically from the cytoplasm to the hyphal tip, where it interacts with MoSec4, a Rab GTPase involved in secretory transport, hyphal growth and fungal pathogenicity. Our studies indicate that class V myosin and its translocation are tightly coupled with hyphal growth, asexual reproduction, appressorium function and pathogenicity in the rice blast fungus.     

MoCDC14 is important for septation during conidiation and appressorium formation in Magnaporthe oryzae

As a typical foliar pathogen, appressorium formation and penetration are critical steps in the infection cycle of Magnaporthe oryzae. Because appressorium formation and penetration are closely co‐regulated with the cell cycle, and Cdc14 phosphatases have an antagonistic relationship with cyclin‐dependent kinases (CDKs) on proteins related to mitotic exit and cytokinesis, in this study, we functionally characterized the MoCDC14 gene in M. oryzae. The Mocdc14 deletion mutant showed significantly reduced growth rate and conidiation. It was also defective in septum formation and nuclear distribution. Septation was irregular in Mocdc14 hyphae and hyphal compartments became multi‐nucleate. Mutant conidia often showed incomplete septa or lacked any septum. During appressorium formation, the septum delimiting appressoria from the rest of the germ tubes was often formed far away from the neck of the appressoria or not formed at all. Unlike the wild‐type, some mutant appressoria had more than one nucleus at 24 h. In addition to appressoria, melanization occurred on parts of the germ tubes and conidia, depending on the irregular position of the appressorium‐delimiting septum. The Mocdc14 mutant was also defective in glycogen degradation during appressorium formation and appressorial penetration of intact plant cells. Similar defects in septum formation, melanization and penetration were observed with appressorium‐like structures formed at hyphal tips in the Mocdc14 mutant. Often a long fragment of mutant hyphae was melanized, together with the apical appressorium‐like structures. These results indicate that MoCDC14 plays a critical role in septation, nuclear distribution and pathogenesis in M. oryzae, and correct septum formation during conidiogenesis and appressorium formation requires the MoCdc14 phosphatase.     

The Supernatant of a Conidial Suspension of Magnaporthe oryzae Contains a Factor that Promotes the Infection of Rice Plants

Many factors produced by the pathogen Magnaporthe oryzae enhance its ability to infect rice. We found a novel infection-promoting activity in the supernatant of a conidia suspension (SCS) of M . oryzae . The addition of SCS promoted the invasion of excised rice leaf sheaths by infectious hyphae. The activity was heat-stable and was found in SCSs from five virulent and three avirulent isolates of M . oryzae on the rice cv. Nipponbare ( Pia ). The effect was exclusively detected in compatible interactions. The infection of rice plants by non-rice blast fungi was not enhanced by SCS. These results suggest that SCS includes a heat-stable factor(s) that promotes M . oryzae infection during compatible interactions.     

Resistance-related physiological response of rice leaves to the compound stress of enhanced UV-B radiation and Magnaporthe oryzae

An enhanced UV-B radiation (5.0?kJ?m^?2) was supplied before, during, and after Magnaporthe oryzae infection. The effects of single and compound stress of the UV-B radiation and M. oryzae on the resistance physiology and gene expression of rice leaves were examined. Results revealed that UV-B radiation given before M. oryzae infection (UV-B?→?M.) significantly increased the pathogenesis-related proteins (PRs) activities of phenylalanine ammonialyase (PAL), lipoxygenase (LOX), chitinase (CHT), and β-1,3-glucanase, the resistance-related substances (flavonoids and total phenols) content, and resistance-related genes (OsPAL and OsCHT) expression, thereby improving the disease resistance of rice leaves. Simultaneous exposure to UV-B radiation and M. oryzae (UV-B/M.) significantly increased the OsLOX2 expression and the PRs activities. Exposure to UV-B radiation after M. oryzae infection (M.?→?UV-B) decreased the flavonoid content, did not improve the PRs activity, and increased OsLOX2 expression. Compound treatments of UV-B?→?M., UV-B/M., and M.?→?UV-B reduced the disease index by 62.3%, 40.2%, and 26.6%, respectively, indicating UV-B radiation inhibited the occurrence of M. oryzae disease, but its inhibitory effect weakened when it was provided after M. oryzae infection. Hence, rice responded to the compound stress of UV-B radiation and M. oryzae through a resistance-related physiological mechanism associated with the sequence of stress occurrence.     

MoWhi2 regulates appressorium formation and pathogenicity via the MoTor signalling pathway in Magnaporthe oryzae

Magnaporthe oryzae causes rice blast disease, which seriously threatens the safety of food production. Understanding the mechanism of appressorium formation, which is one of the key steps for successful infection by M. oryzae, is helpful to formulate effective control strategies of rice blast. In this study, we identified MoWhi2, the homolog of Saccharomyces cerevisiae Whi2 (Whisky2), as an important regulator that controls appressorium formation in M. oryzae. When MoWHI2 was disrupted, multiple appressoria were formed by one conidium and pathogenicity was significantly reduced. A putative phosphatase, MoPsr1, was identified to interact with MoWhi2 using a yeast two-hybridization screening assay. The knockout mutant ΔMopsr1 displayed similar phenotypes to the ΔMowhi2 strain. Both the ΔMowhi2 and ΔMopsr1 mutants could form appressoria on a hydrophilic surface with cAMP levels increasing in comparison with the wild type (WT). The conidia of ΔMowhi2 and ΔMopsr1 formed a single appressorium per conidium, similar to WT, when the target of rapamycin (TOR) inhibitor rapamycin was present. In addition, compared with WT, the expression levels of MoTOR and the MoTor signalling activation marker gene MoRS3 were increased, suggesting that inappropriate activation of the MoTor signalling pathway is one of the important reasons for the defects in appressorium formation in the ΔMowhi2 and ΔMopsr1 strains. Our results provide insights into MoWhi2 and MoPsr1-mediated appressorium development and pathogenicity by regulating cAMP levels and the activation of MoTor signalling in M. oryzae.     

Heterologous production,purification, and immunodetection of Magnaporthe oryzae avirulence protein AVR-Pia

The avirulence gene AVR-Pia of Magnaporthe oryzae, which induces a hypersensitive reaction in rice cultivars containing the resistance gene Pia, was expressed in Escherichia coli. AVR-Pia protein was collected as inclusion bodies, denatured, and refolded. Finally, recombinant AVR-Pia (rAVR-Pia) protein was purified by column chromatography. Infiltration of rAVR-Pia triggered cell browning in the leaves of rice cultivar Aichiasahi (Pia), with accumulation of H₂O₂ and induction of PR1a expression in rice. On the other hand, these reactions were not observed in Shin-2 (pia) leaves after the same treatment. This observation indicated that rAVR-Pia had the function of an avirulence protein. rAVR-Pia was used for immunization of a rabbit, and anti-AVR-Pia antiserum was prepared. The specificity of this antibody was appraised by detecting native AVR-Pia in the inoculated leaf sheath extract using Western blotting in combination with immunoprecipitation. Native AVR-Pia was successfully detected, and its molecular weight was estimated to be 7.4?kDa, indicating signal peptide cleavage. Additionally, secreted native AVR-Pia was quantified as 3.7?ng/g rice sheath.