Evidence for Biotrophic Lifestyle and Biocontrol Potential of Dark Septate Endophyte Harpophora oryzae to Rice Blast Disease

The mutualism pattern of the dark septate endophyte (DSE) Harpophora oryzae in rice roots and its biocontrol potential in rice blast disease caused by Magnaporthe oryzae were investigated. Fluorescent protein-expressing H. oryzae was used to monitor the colonization pattern. Hyphae invaded from the epidermis to the inner cortex, but not into the root stele. Fungal colonization increased with root tissue maturation, showing no colonization in the meristematic zone, slight colonization in the elongation zone, and heavy colonization in the differentiation zone. H. oryzae adopted a biotrophic lifestyle in roots accompanied by programmed cell death. Real-time PCR facilitated the accurate quantification of fungal growth and the respective plant response. The biocontrol potential of H. oryzae was visualized by inoculation with eGFP-tagged M. oryzae in rice. H. oryzae protected rice from M. oryzae root invasion by the accumulation of H₂O₂ and elevated antioxidative capacity. H. oryzae also induced systemic resistance against rice blast. This systemic resistance was mediated by the OsWRKY45-dependent salicylic acid (SA) signaling pathway, as indicated by the strongly upregulated expression of OsWRKY45. The colonization pattern of H. oryzae was consistent with the typical characteristics of DSEs. H. oryzae enhanced local resistance by reactive oxygen species (ROS) and high antioxidative level and induced OsWRKY45-dependent SA-mediated systemic resistance against rice blast.     

五个WRKY转录因子在水稻叶片生长和抗病反应中的表达研究

WRKY是植物基因组中最大的转录因子家族之一,它们在抗病及其他信号转导途径中发挥着重要的调控作用．为了解水稻WRKY的功能,我们选择了5个WRKY转录因子,用免疫印迹技术调查了它们在水稻叶片生长和在Xa21介导的白叶枯病抗性反应中的表达丰度变化．结果表明,OsWRKY13、23和71在叶片中表达,且随叶片生长而逐步增加,至成熟期略有下降,但在叶片中检测不到OsWRKY45和OsWRKY53的表达信号．在Xa21介导的白叶枯病抗性反应中,OsWRKY45、53和71均受诱导表达,而OsWRKY13和 OsWRKY23蛋白质的表达没有可见的变化．进一步比较OsWRKY45、OsWRKY53和OsWRKY71在抗、感和对照(Mock)反应中的表达,发现它们在抗、感反应中均发生相似变化．上述结果说明,OsWRKY13和OsWRKY23可能在叶片正常生长过程中发挥作用,OsWRKY45和OsWRKY53可能在水稻-白叶枯病菌互作过程中发挥作用,而OsWRKY71在二种条件下均有功能．     

Abscisic Acid Promotes Susceptibility to the Rice Leaf Blight Pathogen Xanthomonas oryzae pv oryzae by Suppressing Salicylic Acid-Mediated Defenses

The plant hormone abscisic acid (ABA) is involved in a wide variety of plant processes, including the initiation of stress-adaptive responses to various environmental cues. Recently, ABA also emerged as a central factor in the regulation and integration of plant immune responses, although little is known about the underlying mechanisms. Aiming to advance our understanding of ABA-modulated disease resistance, we have analyzed the impact, dynamics and interrelationship of ABA and the classic defense hormone salicylic acid (SA) during progression of rice infection by the leaf blight pathogen Xanthomonas oryzae pv. oryzae (Xoo). Consistent with ABA negatively regulating resistance to Xoo, we found that exogenously administered ABA renders rice hypersusceptible to infection, whereas chemical and genetic disruption of ABA biosynthesis and signaling, respectively, led to enhanced Xoo resistance. In addition, we found successful Xoo infection to be associated with extensive reprogramming of ABA biosynthesis and response genes, suggesting that ABA functions as a virulence factor for Xoo. Interestingly, several lines of evidence indicate that this immune-suppressive effect of ABA is due at least in part to suppression of SA-mediated defenses that normally serve to limit pathogen growth. Resistance induced by the ABA biosynthesis inhibitor fluridone, however, appears to operate in a SA-independent manner and is likely due to induction of non-specific physiological stress. Collectively, our findings favor a scenario whereby virulent Xoo hijacks the rice ABA machinery to cause disease and highlight the importance of ABA and its crosstalk with SA in shaping the outcome of rice-Xoo interactions.     

A Rice Gene of De Novo Origin Negatively Regulates Pathogen-Induced Defense Response

How defense genes originated with the evolution of their specific pathogen-responsive traits remains an important problem. It is generally known that a form of duplication can generate new genes, suggesting that a new gene usually evolves from an ancestral gene. However, we show that a new defense gene in plants may evolve by de novo origination, resulting in sophisticated disease-resistant functions in rice. Analyses of gene evolution showed that this new gene, OsDR10, had homologs only in the closest relative, Leersia genus, but not other subfamilies of the grass family; therefore, it is a rice tribe-specific gene that may have originated de novo in the tribe. We further show that this gene may evolve a highly conservative rice-specific function that contributes to the regulation difference between rice and other plant species in response to pathogen infections. Biologic analyses including gene silencing, pathologic analysis, and mutant characterization by transformation showed that the OsDR10-suppressed plants enhanced resistance to a broad spectrum of Xanthomonas oryzae pv. oryzae strains, which cause bacterial blight disease. This enhanced disease resistance was accompanied by increased accumulation of endogenous salicylic acid (SA) and suppressed accumulation of endogenous jasmonic acid (JA) as well as modified expression of a subset of defense-responsive genes functioning both upstream and downstream of SA and JA. These data and analyses provide fresh insights into the new biologic and evolutionary processes of a de novo gene recruited rapidly.     

The C-terminal region of OsWRKY30 is sufficient to confer enhanced resistance to pathogen and activate the expression of defense-related genes

WRKY proteins are known to play major roles in defense signaling. We identified a pathogen-inducible and SA-inducible OsWRKY30. Our cDNA clone encodes the C-terminal region (CTR) of OsWRKY30. CTR-OsWRKY30 includes the C-terminal WRKY domain and nuclear localization sequence. CTR-OsWRKY30 was sufficient to bind W-box sequences (TTGACC/T). Over-expression of the CTR-OsWRKY30 resulted in enhanced resistance to pathogens in Arabidopsis and rice. Defense-related genes were constitutively expressed in transgenic Arabidopsis and rice over-expressing CTR-OsWRKY30. Based on promoter transient assays, CTR-OsWRKY30 is sufficient to activate OsPR10a promoter as much as full length OsWRKY30. Taken together, CTR-OsWRKY30 positively regulates defense signaling, thereby resulting in enhanced resistance to pathogens.     

Functional complementation of rice blast resistance gene Pi-k h (Pi54) conferring resistance to diverse strains of Magnaporthe oryzae

Blast disease of rice, caused by Magnaporthe oryzae is an explosive disease that can spread rapidly in conducive conditions. R-gene mediated resistance offers an environmentally sustainable solution for management of this important disease of rice. We have earlier identified a unique R-gene of rice, on chromosome 11 of Oryza sativa ssp. indica cultivar Tetep. In this study we report functional validation of the Pi-k ^h (Pi54) gene using complementation assay. The blast resistance candidate gene Pi-k ^h (Pi54) was cloned into a plant transformation vector and the construct was used to transform a japonica cultivar of rice Taipei 309, which is susceptible to M. oryzae. Transgenic lines containing Pi-k ^h (Pi54) gene were found to confer high degree of resistance to diverse isolates of M. oryzae. The callose deposition was analyzed and compared between the transgenic and non-transgenic rice plants and widespread deposition was observed at the infection sites in plants showing incompatible interaction. Successful complementation of Pi-k ^h (Pi54) gene confirmed that the gene is responsible for resistance to M. oryzae in transgenic lines developed during this study. Expression analysis of the gene in resistant plants revealed that the gene is pathogen inducible in nature and is not expressed constitutively. Detection of callose deposition in resistant plants containing Pi-k ^h (Pi54) gene implicates its involvement in the initiation of defense response cascade.