Project description:Glufosinate is an important and widely used non-selective herbicide active on a wide range of plant species. Evolution of resistance to glufosinate in weedy plant species (including the global weed Eleusine indica) is underway. Here, we established the molecular basis of target site glufosinate resistance in Eleusine indica. Full-length E. indica glutamine synthetase (GS) iso-genes (EiGS1-1, 1-2, 1-3, and EiGS2) were cloned, and expression of EiGS1-1 and EiGS1-2 was higher than that of EiGS2. A novel point mutation resulting in a Ser59Gly substitution in EiGS1-1 was identified in glufosinate-resistant plants. Rice calli and seedlings transformed with the mutant EiGS1-1 gene were resistant to glufosinate. Purified mutant EiGS1-1 expressed in yeast was more tolerant to glufosinate than the wild-type variant. These transgenic results correlate with a more glufosinate-resistant GS in the crude tissue extract of resistant versus susceptible E. indica plants. Structural modelling of the mutant EiGS1-1 revealed that Ser59 is not directly involved in glufosinate binding but is in contact with some important binding residues (e.g. Glu297) and especially with Asp56 that forms an intratoroidal contact interface. Importantly, the same Ser59Gly mutation was also found in geographically isolated glufosinate-resistant populations from Malaysia and China, suggesting parallel evolution of this resistance mutation.
Project description:Rice sheath blight (ShB) is a devastating disease that severely threatens rice production worldwide. Induction of cell death represents a key step during infection by the ShB pathogen Rhizoctonia solani. Nonetheless, the underlying mechanisms remain largely unclear. In the present study, we identified a rice transcription factor, OsERF65, that negatively regulates resistance to ShB by suppressing cell death. OsERF65 was significantly upregulated by R. solani infection in susceptible cultivar Lemont and was highly expressed in the leaf sheath. Overexpression of OsERF65 (OsERF65OE) decreased rice resistance, while the knockout mutant (oserf65) exhibited significantly increased resistance against ShB. The transcriptome assay revealed that OsERF65 repressed the expression of peroxidase genes after R. solani infection. The antioxidative enzyme activity was significantly increased in oserf65 plants but reduced in OsERF65OE plants. Consistently, hydrogen peroxide content was apparently reduced in oserf65 plants but accumulated in OsERF65OE plants. OsERF65 directly bound to the GCC box in the promoter regions of four peroxidase genes and suppressed their transcription, reducing the ability to scavenge reactive oxygen species (ROS). The oserf65 mutant exhibited a slight decrease in plant height but increased grain yield. Overall, our results revealed an undocumented role of OsERF65 that acts as a crucial regulator of rice resistance to R. solani and a potential target for improving both ShB resistance and rice yield.
Project description:Catalases (CATs) are important self-originating enzymes and are involved in many of the biological functions of plants. Multiple forms of CATs suggest their versatile role in lesion mimic mutants (LMMs), H2O2 homeostasis and abiotic and biotic stress tolerance. In the current study, we identified a large lesion mimic mutant9428 (llm9428) from Ethyl-methane-sulfonate (EMS) mutagenized population. The llm9428 showed a typical phenotype of LMMs including decreased agronomic yield traits. The histochemical assays showed decreased cell viability and increased reactive oxygen species (ROS) in the leaves of llm9428 compared to its wild type (WT). The llm9428 showed enhanced blast disease resistance and increased relative expression of pathogenesis-related (PR) genes. Studies of the sub-cellular structure of the leaf and quantification of starch contents revealed a significant decrease in starch granule formation in llm9428. Genetic analysis revealed a single nucleotide change (C > T) that altered an amino acid (Ala > Val) in the candidate gene (Os03g0131200) encoding a CATALASE C in llm9428. CRISPR-Cas9 targetted knockout lines of LLM9428/OsCATC showed the phenotype of LMMs and reduced starch metabolism. Taken together, the current study results revealed a novel role of OsCATC in starch metabolism in addition to validating previously studied functions of CATs.
Project description:Previous research has shown that the pathogenicity and appressorium development of Magnaporthe oryzae can be inhibited by the ATP synthase subunit beta (Atp2) present in the photosynthetic bacterium Rhodopseudomonas palustris. In the present study, transgenic plants overexpressing the ATP2 gene were generated via genetic transformation in the Zhonghua11 (ZH11) genetic background. We compared the blast resistance and immune response of ATP2-overexpressing lines and wild-type plants. The expression of the Atp2 protein and the physiology, biochemistry, and growth traits of the mutant plants were also examined. The results showed that, compared with the wild-type plant ZH11, transgenic rice plants heterologously expressing ATP2 had no significant defects in agronomic traits, but the disease lesions caused by the rice blast fungus were significantly reduced. When infected by the rice blast fungus, the transgenic rice plants exhibited stronger antioxidant enzyme activity and a greater ratio of chlorophyll a to chlorophyll b. Furthermore, the immune response was triggered stronger in transgenic rice, especially the increase in reactive oxygen species (ROS), was more strongly triggered in plants. In summary, the expression of ATP2 as an antifungal protein in rice could improve the ability of rice to resist rice blast.
Project description:CRISPR crops carrying a mutation in susceptibility (S) genes provide an effective strategy for controlling plant disease, because they could be 'transgene-free' and commonly have more broad-spectrum and durable type of resistance. Despite their importance, CRISPR/Cas9-mediated editing of S genes for engineering resistance to plant-parasitic nematode (PPN) disease has not been reported. In this study, we employed the CRISPR/Cas9 system to specifically induce targeted mutagenesis of the S gene rice copper metallochaperone heavy metal-associated plant protein 04 (OsHPP04), and successfully obtained genetically stable homozygous rice mutants with or without transgenic elements. These mutants confer enhanced resistance to the rice root-knot nematode (Meloidogyne graminicola), a major plant pathogenic nematode in rice agriculture. Moreover, the plant immune responses triggered by flg22, including reactive oxygen species burst, defence-related genes expression and callose deposition, were enhanced in the 'transgene-free' homozygous mutants. Analysis of rice growth and agronomic traits of two independent mutants showed that there are no obvious differences between wild-type plants and mutants. These findings suggest that OsHPP04 may be an S gene as a negative regulator of host immunity and genetic modification of S genes through the CRISPR/Cas9 technology can be used as a powerful tool to generate PPN resistant plant varieties.
Project description:Sheath blight (ShB), caused by Rhizoctonia solani, is a highly destructive disease in many crops worldwide and no major resistance genes are available. Here, we identified a sbr1 (sheath blight resistance 1) rice mutant, which shows enhanced ShB resistance and maintains wildtype agronomic traits including yield, but carries an undesired stay-green phenotype. Through map-based cloning and transgenic validation, we found that an insertion disrupting the Stay-Green (OsSGR) gene is responsible for sbr1 phenotypes. Mechanistically, the sbr1/Ossgr mutants reduce the expression of most OsCKX genes, which function in cytokinin (CK) degradation, to accumulate CK leading to ShB resistance. Importantly, knockout of OsCKX7, predominantly expressed in the leaf sheath and highly induced by R. solani, significantly enhances ShB resistance without stay-green phenotype nor yield penalty, showing high application potential. Thus, our study reveals novel insights that OsSGR and cytokinin play key roles in rice-R. solani interaction and generates a valuable ShB-resistant germplasm.
Project description:Polyamines are involved not only in fundamental cellular processes such as growth, differentiation, and morphogenesis, but also in various environmental stresses. We demonstrated that spermidine, a polyamine, confers resistance to rice blast accompanied by the up-regulation of marker genes for the salicylic acid-mediated signaling pathway PR1b and PBZ1 and of phytoalexin biosynthesis genes CPS4 and NOMT. This is the first report about the involvement of spermidine in rice disease resistance.
Project description:Although molecular hydrogen (H2) has potential therapeutic effects in animals, whether or how this gas functions in plant disease resistance has not yet been elucidated. Here, after rice stripe virus (RSV) infection, H2 production was pronouncedly stimulated in Zhendao 88, a resistant rice variety, compared to that in a susceptible variety (Wuyujing No.3). External H2 supply remarkably reduced the disease symptoms and RSV coat protein (CP) levels, especially in Wuyujing No.3. The above responses were abolished by the pharmacological inhibition of H2 production. The transgenic Arabidopsis plants overexpressing a hydrogenase gene from Chlamydomonas reinhardtii also improved plant resistance. In the presence of H2, the transcription levels of salicylic acid (SA) synthetic genes were stimulated, and the activity of SA glucosyltransferases was suppressed, thus facilitating SA accumulation. Genetic evidence revealed that two SA synthetic mutants of Arabidopsis (sid2-2 and pad4) were more susceptible to RSV than the wild type (WT). The treatments with H2 failed to improve the resistance to RSV in two SA synthetic mutants. The above results indicated that H2 enhances rice resistance to RSV infection possibly through the SA-dependent pathway. This study might open a new window for applying the H2-based approach to improve plant disease resistance. IMPORTANCE Although molecular hydrogen has potential therapeutic effects in animals, whether or how this gas functions in plant disease resistance has not yet been elucidated. RSV was considered the most devastating plant virus in rice, since it could cause severe losses in field production. This disease was thus selected as a classical model to explore the interrelationship between molecular hydrogen and plant pathogen resistance. In this study, we discovered that both exogenous and endogenous H2 could enhance plant resistance against Rice stripe virus infection by regulating salicylic acid signaling. Compared with some frequently used agrochemicals, H2 is almost nontoxic. We hope that the findings presented here will serve as an opportunity for the scientific community to push hydrogen-based agriculture forward.