SOBIR1 contributes to non-host resistance to Magnaporthe oryzae in Arabidopsis

The rate of entry of Magnaporthe oryzae into Arabidopsis pen2 sobir1 plants was significantly higher than that into pen2 plants. The length of the infection hyphae in pen2 sobir1 plants was significantly longer than that in pen2 plants. These results suggest that SOBIR1 is involved in both penetration and post-penetration resistance to M. oryzae in Arabidopsis.     

Penetration and Post-Penetration Resistance to Magnaporthe oryzae in Arabidopsis

The rate of entry of Magnaporthe oryzae into the Arabidopsis pen2 quintuple (pen2 NahG pmr5 agb1 mlo2) mutant was significantly higher than those into the pen2 quadruple (pen2 NahG pmr5 agb1 and pen2 NahG pmr5 mlo2) mutants. The lengths of the infection hyphae in the pen2 quintuple mutant were intermediate between the pen2 quadruple mutants. These results suggest that different genetic networks, consisting of PEN2, PMR5, AGB1, and MLO2, control penetration and post-penetration resistance to M. oryzae in Arabidopsis.     

SOBIR1 and AGB1 independently contribute to nonhost resistance to Pyricularia oryzae (syn. Magnaporthe oryzae) in Arabidopsis thaliana

ABSTRACT

Rice blast caused by Pyricularia oryzae (syn. Magnaporthe oryzae) is a disease devastating to rice. We have studied the Arabidopsis-P. oryzae pathosystem as a model system for nonhost resistance (NHR) and found that SOBIR1, but not BAK1, is a positive regulator of NHR to P. oryzae in Arabidopsis. AGB1 is also involved in NHR. However, the genetic interactions between SOBIR1, BAK1, and AGB1 are uncharacterized. In this study, we delineated the genetic interactions between SOBIR1, BAK1, and AGB1 in NHR to P. oryzae in Arabidopsis and found SOBIR1 and AGB1 independently control NHR to P. oryzae in Arabidopsis pen2-1 mutant plants. Furthermore, XLG2, but not TMM, has a positive role in penetration resistance to P. oryzae in Arabidopsis pen2-1 mutant plants. Our study characterized genetic interactions in Arabidopsis NHR.

Abbreviations: PRR: pattern recognition receptor, RLK: receptor-like kinase, RLP: receptor-like protein, BAK1: BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1, BIR1: BAK1-INTERACTING RECEPTOR-LIKE KINASE 1, SOBIR1: SUPPRESSOR OF BIR1-1-1, AGB1: ARABIDOPSIS G PROTEIN ß-SUBUNIT 1, XLG2: EXTRA-LARGE G PROTEIN 2     

Endophytic fungus Falciphora oryzae promotes lateral root growth by producing indole derivatives after sensing plant signals

The endophytic fungus Falciphora oryzae was initially isolated from wild rice (Oryza granulata) and colonizes many crop species and promotes plant growth. However, the molecular mechanisms underlying F. oryzae-mediated growth promotion are still unknown. We found that F. oryzae was able to colonize Arabidopsis thaliana. The most dramatic change after F. oryzae inoculation was observed in the root architecture, as evidenced by increased lateral root growth but reduced primary root length, similar to the effect of auxin, a significant plant growth hormone. The expression of genes responsible for auxin biosynthesis, transport, and signalling was regulated in Arabidopsis roots after F. oryzae cocultivation. Indole derivatives were detected at significantly higher levels in liquid media after cocultivation compared with separate cultivation of Arabidopsis and F. oryzae. Consistently, the expression of indole biosynthetic genes was highly upregulated in F. oryzae upon treatment with Arabidopsis exudates. Global analysis of Arabidopsis gene expression at the early stage after F. oryzae cocultivation suggested that signals were exchanged to initiate Arabidopsis–F. oryzae interactions. All these results suggest that signalling molecules from Arabidopsis roots are perceived by F. oryzae and induce the biosynthesis of indole derivatives in F. oryzae, consequently stimulating Arabidopsis lateral root growth.     

Rice ubiquitin-conjugating enzyme OsUBC26 is essential for immunity to the blast fungus Magnaporthe oryzae

The functions of ubiquitin-conjugating enzymes (E2) in plant immunity are not well understood. In this study, OsUBC26, a rice ubiquitin-conjugating enzyme, was characterized in the defence against Magnaporthe oryzae. The expression of OsUBC26 was induced by M. oryzae inoculation and methyl jasmonate treatment. Both RNA interference lines and CRISPR/Cas9 null mutants of OsUBC26 reduced rice resistance to M. oryzae. WRKY45 was down-regulated in OsUBC26 null mutants. In vitro E2 activity assay indicated that OsUBC26 is an active ubiquitin-conjugating enzyme. Yeast two-hybrid assays using OsUBC26 as bait identified the RING-type E3 ligase UCIP2 as an interacting protein. Coimmunoprecipitation assays confirmed the interaction between OsUBC26 and UCIP2. The CRISPR/Cas9 mutants of UCIP2 also showed compromised resistance to M. oryzae. Yeast two-hybrid screening using UCIP2 as bait revealed that APIP6 is a binding partner of UCIP2. Moreover, OsUBC26 working with APIP6 ubiquitinateds AvrPiz-t, an avirulence effector of M. oryzae, and OsUBC26 null mutation impaired the proteasome degradation of AvrPiz-t in rice cells. In summary, OsUBC26 plays important roles in rice disease resistance by regulating WRKY45 expression and working with E3 ligases such as APIP6 to counteract the effector protein AvrPiz-t from M. oryzae.     

Herbivore-induced rice resistance against rice blast mediated by salicylic acid

In agro-ecosystems,plants are important mediators of interactions between their associated herbivorous insects and microbes,and any change in plants induced by one species may lead to cascading effects on interactions with other species.Often,such effects are regulated by phytohormones such as jasmonic acid(JA)and salicylic acid(SA).Here,we investigated the tripartite interactions among rice plants,three insect herbivores(Chilo suppressalis,Cnaphalocrocis medinalis or Nilapai-vata lugens),and the causal agent of rice blast disease,the fungus Magnaporthe oryzae.We found that pre-infestation of rice by C.suppressalis or N.lugens but not by C.medinalis conferred resistance to M.oryzae.For C.suppressalis and N.lugens,insect infestation without fungal inoculation induced the accumulation of both JA and SA in rice leaves.In contrast,infestation by C.medinalis increased JA levels but reduced SA levels.The exogenous application of SA but not of JA conferred resistance against M.oryzae.These results suggest that preinfestation by C suppressalis or N.lugens conferred resistance against M.oryzae by increasing SA accumulation.These findings enhance our understanding of the interactions among rice plant,insects and pathogens,and provide valuable information for developing an ecologically sound strategy for controlling rice blast.     

Intercellular Production of Tamavidin 1, a Biotin-Binding Protein from Tamogitake Mushroom,Confers Resistance to the Blast Fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> in Transgenic Rice

The blast fungus Magnaporthe oryzae, one of the most devastating rice pathogens in the world, shows biotin-dependent growth. We have developed a strategy for creating disease resistance to M. oryzae whereby intercellular production of tamavidin 1, a biotin-binding protein from Pleurotus cornucopiae occurs in transgenic rice plants. The gene that encodes tamavidin 1, fused to the sequence for a secretion signal peptide derived from rice chitinase gene, was connected to the Cauliflower mosaic virus 35S promoter, and the resultant construct was introduced into rice. The tamavidin 1 was accumulated at levels of 0.1–0.2% of total soluble leaf proteins in the transgenic rice and it was localized in the intercellular space of rice leaves. The tamavidin 1 purified from the transgenic rice was active, it bound to biotin and inhibited in vitro growth of M. oryzae by causing biotin deficiency. The transgenic rice plants showed a significant resistance to M. oryzae. This study shows the possibility of a new strategy to engineer disease resistance in higher plants by taking advantage of a pathogen’s auxotrophy.     

Expression of a bacterial chitosanase in rice plants improves disease resistance to the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Plant fungal pathogens change their cell wall components during the infection process to avoid degradation by host lytic enzymes, and conversion of the cell wall chitin to chitosan is likely to be one infection strategy of pathogens. Thus, introduction of chitosan-degradation activity into plants is expected to improve fungal disease resistance. Chitosanase has been found in bacteria and fungi, but not in higher plants. Here, we demonstrate that chitosanase, Cho1, from Bacillus circulans MH-K1 has antifungal activity against the rice blast fungus Magnaporthe oryzae. Introduction of the cho1 gene conferred chitosanase activity to rice cells. Transgenic rice plants expressing Cho1 designed to be localized in the apoplast showed increased resistance to M. oryzae accompanied by increased generation of hydrogen peroxide in the infected epidermal cells. These results strongly suggest that chitosan exists in the enzyme-accessible surface of M. oryzae during the infection process and that the enhancement of disease resistance is attributable to the antifungal activity of the secreted Cho1 and to increased elicitation of the host defense response.     

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.     

MoOpy2 is essential for fungal development,pathogenicity, and autophagy in Magnaporthe oryzae

The development and pathogenicity of the fungus Magnaporthe oryzae, the causal agent of destructive rice blast disease, require it to perceive external environmental signals. Opy2, an overproduction-induced pheromone-resistant protein 2, is a crucial protein for sensing external signals in Saccharomyces cerevisiae. However, the biological functions of the homologue of Opy2 in M. oryzae are unclear. In this study, we identified that MoOPY2 is involved in fungal development, pathogenicity, and autophagy in M. oryzae. Deletion of MoOPY2 resulted in pleiotropic defects in hyphal growth, conidiation, germ tube extension, appressorium formation, appressorium turgor generation, and invasive growth, therefore leading to attenuated pathogenicity. Furthermore, MoOpy2 participates in the Osm1 MAPK pathway and the Mps1 MAPK pathway by interacting with the adaptor protein Mst50. The interaction sites of Mst50 and MoOpy2 colocalized with the autophagic marker protein MoAtg8 in the preautophagosomal structure sites (PAS). Notably, the ΔMoopy2 mutant caused cumulative MoAtg8 lipidation and rapid GFP-MoAtg8 degradation in response to nitrogen starvation, showing that MoOpy2 is involved in the negative regulation of autophagy activity. Taken together, our study revealed that MoOpy2 of M. oryzae plays an essential role in the orchestration of fungal development, appressorium penetration, autophagy and pathogenesis.     

Overexpression of a rice heme activator protein gene (OsHAP2E) confers resistance to pathogens,salinity and drought,and increases photosynthesis and tiller number

Heme activator protein (HAP), also known as nuclear factor Y or CCAAT binding factor (HAP/NF‐Y/CBF), has important functions in regulating plant growth, development and stress responses. The expression of rice HAP gene (OsHAP2E) was induced by probenazole (PBZ), a chemical inducer of disease resistance. To characterize the gene, the chimeric gene (OsHAP2E::GUS) engineered to carry the structural gene encoding β‐glucuronidase (GUS) driven by the promoter from OsHAP2E was introduced into rice. The transgenic lines of OsHAP2Ein::GUS with the intron showed high GUS activity in the wounds and surrounding tissues. When treated by salicylic acid (SA), isonicotinic acid (INA), abscisic acid (ABA) and hydrogen peroxide (H₂O₂), the lines showed GUS activity exclusively in vascular tissues and mesophyll cells. This activity was enhanced after inoculation with Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae. The OsHAP2E expression level was also induced after inoculation of rice with M. oryzae and X. oryzae pv. oryzae and after treatment with SA, INA, ABA and H₂O_2, respectively. We further produced transgenic rice overexpressing OsHAP2E. These lines conferred resistance to M. oryzae or X. oryzae pv. oryzae and to salinity and drought. Furthermore, they showed a higher photosynthetic rate and an increased number of tillers. Microarray analysis showed up‐regulation of defence‐related genes. These results suggest that this gene could contribute to conferring biotic and abiotic resistances and increasing photosynthesis and tiller numbers.     

The rice terpene synthase gene OsTPS19 functions as an (S)‐limonene synthase in planta,and its overexpression leads to enhanced resistance to the blast fungus Magnaporthe oryzae

Rice blast disease, caused by the fungus Magnaporthe oryzae, is the most devastating disease of rice. In our ongoing characterization of the defence mechanisms of rice plants against M. oryzae, a terpene synthase gene OsTPS19 was identified as a candidate defence gene. Here, we report the functional characterization of OsTPS19, which is up‐regulated by M. oryzae infection. Overexpression of OsTPS19 in rice plants enhanced resistance against M. oryzae, while OsTPS19 RNAi lines were more susceptible to the pathogen. Metabolic analysis revealed that the production of a monoterpene (S)‐limonene was increased and decreased in OsTPS19 overexpression and RNAi lines, respectively, suggesting that OsTPS19 functions as a limonene synthase in planta. This notion was further supported by in vitro enzyme assays with recombinant OsTPS19, in which OsTPS19 had both sesquiterpene activity and monoterpene synthase activity, with limonene as a major product. Furthermore, in a subcellular localization experiment, OsTPS19 was localized in plastids. OsTPS19 has a highly homologous paralog, OsTPS20, which likely resulted from a recent gene duplication event. We found that the variation in OsTPS19 and OsTPS20 enzyme activities was determined by a single amino acid in the active site cavity. The expression of OsTPS20 was not affected by M. oryzae infection. This indicates functional divergence of OsTPS19 and OsTPS20. Lastly, (S)‐limonene inhibited the germination of M. oryzae spores in vitro. OsTPS19 was determined to function as an (S)‐limonene synthase in rice and plays a role in defence against M. oryzae, at least partly, by inhibiting spore germination.     

Autophagy vitalizes the pathogenicity of pathogenic fungi

Plant pathogenic fungi utilize a series of complex infection structures, in particular the appressorium, to gain entry to and colonize plant tissue. As a consequence of the accumulation of huge quantities of glycerol in the cell the appressorium generates immense intracellular turgor pressure allowing the penetration peg of the appressorium to penetrate the leaf cuticle. Autophagic processes are ubiquitous in eukaryotic cells and facilitate the bulk degradation of macromolecules and organelles. The study of autophagic processes has been extended from the model yeast Saccharomyces cerevisiae to pathogenic fungi such as the rice blast fungus Magnaporthe oryzae. Significantly, null mutants for the expression of M. oryzae autophagy gene homologs lose their pathogenicity for infection of host plants. Clarification of the functions and network of interactions between the proteins expressed by M. oryzae autophagy genes will lead to a better understanding of the role of autophagy in fungal pathogenesis and help in the development of new strategies for disease control.     

The DnaJ protein OsDjA6 negatively regulates rice innate immunity to the blast fungus Magnaporthe oryzae

Rice blast, caused by Magnaporthe oryzae (synonym: Pyricularia oryzae), severely reduces rice production and grain quality. The molecular mechanism of rice resistance to M. oryzae is not fully understood. In this study, we identified a chaperone DnaJ protein, OsDjA6, which is involved in basal resistance to M. oryzae in rice. The OsDjA6 protein is distributed in the entire rice cell. The expression of OsDjA6 is significantly induced in rice after infection with a compatible isolate. Silencing of OsDjA6 in transgenic rice enhances resistance to M. oryzae and also results in an increased burst of reactive oxygen species after flg22 and chitin treatments. In addition, the expression levels of WRKY45, NPR1 and PR5 are increased in OsDjA6 RNAi plants, indicating that OsDjA6 may mediate resistance by affecting the salicylic acid pathway. Finally, we found that OsDjA6 interacts directly with the E3 ligase OsZFP1 in vitro and in vivo. These results suggest that the DnaJ protein OsDjA6 negatively regulates rice innate immunity, probably via the ubiquitination proteasome degradation pathway.     

A rice gene encoding glycosyl hydrolase plays contrasting roles in immunity depending on the type of pathogens

Because pathogens use diverse infection strategies, plants cannot use one-size-fits-all defence and modulate defence responses based on the nature of pathogens and pathogenicity mechanism. Here, we report that a rice glycoside hydrolase (GH) plays contrasting roles in defence depending on whether a pathogen is hemibiotrophic or necrotrophic. The Arabidopsis thaliana MORE1 (M agnaporthe o ryzae re sistance 1) gene, encoding a member of the GH10 family, is needed for resistance against M. oryzae and Alternaria brassicicola, a fungal pathogen infecting A. thaliana as a necrotroph. Among 13 rice genes homologous to MORE1, 11 genes were induced during the biotrophic or necrotrophic stage of infection by M. oryzae. CRISPR/Cas9-assisted disruption of one of them (OsMORE1a) enhanced resistance against hemibiotrophic pathogens M. oryzae and Xanthomonas oryzae pv. oryzae but increased susceptibility to Cochliobolus miyabeanus, a necrotrophic fungus, suggesting that OsMORE1a acts as a double-edged sword depending on the mode of infection (hemibiotrophic vs. necrotrophic). We characterized molecular and cellular changes caused by the loss of MORE1 and OsMORE1a to understand how these genes participate in modulating defence responses. Although the underlying mechanism of action remains unknown, both genes appear to affect the expression of many defence-related genes. Expression patterns of the GH10 family genes in A. thaliana and rice suggest that other members also participate in pathogen defence.     

Proteomic analysis of rice mutants susceptible to Magnaporthe oryzae

To identify genes involved in rice Pi5-mediated disease resistance to Magnaporthe oryzae, we compared the proteomes of the RIL260 rice strain carrying the Pi5 resistance gene with its susceptible mutants M5465 and M7023. Proteins were extracted from the leaf tissues of both RIL260 and the mutant lines at 0, 24, and 48 h after M. oryzae inoculation and separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified eight proteins that were differently expressed between the resistant and susceptible plants (three down- and five up-regulated proteins in the mutants). The down-regulated proteins included a triosephosphate isomerase (spot no. 2210), a 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (no. 3611), and an unknown protein (no. 4505). In addition, the five up-regulated proteins in the mutants were predicted to be a fructokinase I (no. 313), a glutathione S-transferase (no. 2310), an atpB of chloroplast ATP synthase (no. 3616), an aminopeptidase N (no. 3724), and an unknown protein (no. 308). These results suggest that proteomic analysis of rice susceptible mutants is a useful method for identifying novel proteins involved in resistance to the M. oryzae pathogen.     

2,6‐Dimethoxy‐1,4‐Benzoquinone Enhances Resistance Against the Rice Blast Fungus Magnaporthe oryzae

We investigated the effect of 2,6‐dimethoxy‐1,4‐benzoquinone (DMBQ) on induced resistance to Magnaporthe oryzae in rice. DMBQ concentrations greater than 50 μg/ml inhibited spore germination and appressorium formation in M. oryzae. When rice leaves pretreated with 10 μg/ml DMBQ, which did not show antifungal activity against spore germination and appressorium formation of M. oryzae, were inoculated with M. oryzae spores 5 days after DMBQ pretreatment, blast lesion formation was inhibited compared with control leaves pretreated with distilled water. In addition, infection‐inhibiting activity against M. oryzae was significantly enhanced in rice leaf sheaths pretreated with 10 μg/ml DMBQ. H₂O₂ generation was observed in rice leaves pretreated with DMBQ, and PAL, POX, CHS and PR10a were significantly expressed in these leaves. These results suggested that DMBQ can protect rice from blast disease caused by M. oryzae.     

Osa‐miR1873 fine‐tunes rice immunity against Magnaporthe oryzae and yield traits

MicroRNAs (miRNAs) are known to fine‐tune growth, development, and stress‐induced responses. Osa‐miR1873 is a rice‐specific miRNA targeting LOC_Os05g01790. Here, we show that Osa‐miR1873 fine‐tunes rice immunity against Magnaporthe oryzae and yield traits via LOC_Os05g01790. Osa‐miR1873 was significantly upregulated in a susceptible accession but downregulated in a resistance accession at 24 h post‐inoculation (hpi) of M. oryzae. Overexpressing Osa‐miR1873 enhanced susceptibility to M. oryzae and compromised induction of defense responses. In contrast, blocking Osa‐miR1873 through target mimicry compromised susceptibility to M. oryzae and enhanced induction of defense responses. Altered expression of Osa‐miR1873 also resulted in some defects in yield traits, including grain numbers and seed setting rate. Moreover, overexpression of the target gene LOC_Os05g01790 increased rice blast disease resistance but severely penalized growth and yield. Taken together, we demonstrate that Osa‐miR1873 fine‐tunes the rice immunity‐growth trade‐off via LOC_Os05g01790, and blocking Osa‐miR1873 could improve blast disease resistance without significant yield penalty. Thus, the Osa‐miR1873‐LOC_Os05g01790 regulatory module is valuable in balancing yield traits and blast resistance.