Rice oxalate oxidase gene driven by green tissue‐specific promoter increases tolerance to sheath blight pathogen (Rhizoctonia solani) in transgenic rice

Rice sheath blight, caused by the necrotrophic fungus Rhizoctonia solani, is one of the most devastating and intractable diseases of rice, leading to a significant reduction in rice productivity worldwide. In this article, in order to examine sheath blight resistance, we report the generation of transgenic rice lines overexpressing the rice oxalate oxidase 4 (Osoxo4) gene in a green tissue‐specific manner which breaks down oxalic acid (OA), the pathogenesis factor secreted by R. solani. Transgenic plants showed higher enzyme activity of oxalate oxidase (OxO) than nontransgenic control plants, which was visualized by histochemical assays and sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Transgenic rice leaves were more tolerant than control rice leaves to exogenous OA. Transgenic plants showed a higher level of expression of other defence‐related genes in response to pathogen infection. More importantly, transgenic plants exhibited significantly enhanced durable resistance to R. solani. The overexpression of Osoxo4 in rice did not show any detrimental phenotypic or agronomic effect. Our findings indicate that rice OxO can be utilized effectively in plant genetic manipulation for sheath blight resistance, and possibly for resistance to other diseases caused by necrotrophic fungi, especially those that secrete OA. This is the first report of the expression of defence genes in rice in a green tissue‐specific manner for sheath blight resistance.     

Overexpression of Rice CBS Domain Containing Protein Improves Salinity, Oxidative, and Heavy Metal Tolerance in Transgenic Tobacco

We have recently identified and classified a cystathionine ??-synthase domain containing protein family in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa L.). Based on the microarray and MPSS data, we have suggested their involvement in stress tolerance. In this study, we have characterized a rice protein of unknown function, OsCBSX4. This gene was found to be upregulated under high salinity, heavy metal, and oxidative stresses at seedling stage. Transgenic tobacco plants overexpressing OsCBSX4 exhibited improved tolerance toward salinity, heavy metal, and oxidative stress. This enhanced stress tolerance in transgenic plants could directly be correlated with higher accumulation of OsCBSX4 protein. Transgenic plants could grow and set seeds under continuous presence of 150?mM NaCl. The total seed yield in WT plants was reduced by 80%, while in transgenic plants, it was reduced only by 15?C17%. The transgenic plants accumulated less Na⁺, especially in seeds and maintained higher net photosynthesis rate and Fv/Fm than WT plants under NaCl stress. Transgenic seedlings also accumulated significantly less H₂O₂ as compared to WT under salinity, heavy metal, and oxidative stress. OsCBSX4 overexpressing transgenic plants exhibit higher abiotic stress tolerance than WT plants suggesting its role in abiotic stress tolerance in plants.     

Transgenic indica Rice Expressing ns-LTP-Like Protein Shows Enhanced Resistance to Both Fungal and Bacterial Pathogens

Antimicrobial peptides (AMPs) from plant seeds, known to inhibit pathogen growth have a great potential in developing transgenic plants resistant to disease. Some of the nonspecific-lipid transfer proteins (ns-LTP) that facilitate in vitro transport of lipids, show antimicrobial activity in vitro. Rice seeds also contain ns-LTPs; however, these genes are expressed weakly in seedlings. We have transformed Pusa Basmati 1, an elite indica rice cultivar, with the gene for Ace-AMP1 from Allium cepa, coding for an effective antimicrobial protein homologous to ns-LTPs. The gene for Ace-AMP1 was cloned under an inducible rice phenylalanine ammonia-lyase (PAL) or a constitutive maize ubiquitin (UbI) promoter. Ace-AMP1 was expressed in transgenic lines and secreted in the apoplastic space. Protein extracts from leaves of transgenic plants inhibited three major rice pathogens, Magnaporthe grisea, Rhizoctonia solani and Xanthomonas oryzae, in vitro. Enhanced resistance against these pathogens was observed in in planta assays, and the degree of resistance correlating with the levels of Ace-AMP1 with an average increase in resistance to blast, sheath blight, and bacterial leaf blight disease by 86%, 67%, and 82%, respectively. Importantly, transgenic rice plants, with stable integration and expression of Ace-AMP1, retained their agronomic characteristics while displaying enhanced resistance to both fungal and bacterial pathogens.     

Increased Septoria musiva resistance in transgenic hybrid poplar leaves expressing a wheat oxalate oxidase gene

A cDNA clone of a wheat germin-like oxalate oxidase (OxO) gene regulated by the constitutive CaMV 35S promoter was expressed in a hybrid poplar clone, Populus × euramericana (`Ogy'). Previous studies showed that OxO is likely to play an important role in several aspects of plant development, stress response, and defense against pathogens. In order to study this wheat oxalate oxidase gene in woody plants, the expression of this gene and the functions of the encoded enzyme were examined in vitro and in vivo in transgenic `Ogy'. The enzyme activity in the transformed `Ogy' was visualized by histochemical assays and in SDS-polyacrylamide gels. It was found that the wheat OxO gene is expressed in leaves, stems, and roots of the transgenic `Ogy' plants and the encoded enzyme is able to break down oxalic acid. Transgenic `Ogy' leaves were more tolerant to oxalic acid as well as more effective in increasing the pH in an oxalic acid solution when compared to untransformed controls. In addition, when leaf disks from `Ogy' plants were inoculated with conidia of the poplar pathogenic fungus Septoria musiva, which produces oxalic acid, the OxO-transformed plants were more resistant than the untransformed controls.     

Overexpression of AtNHX5 improves tolerance to both salt and water stress in rice (Oryza sativa L.)

Overexpression of NHX genes has been previously shown to improve salt tolerance of transgenic plants. In this study, transgenic rice plants overexpressing AtNHX5 showed not only high salt tolerance, but also high drought tolerance. Measurements of ion levels indicated that Na⁺ and K⁺ contents were all higher in AtNHX5 overexpressing shoots than in wild type (WT) shoots in high saline conditions. After exposure to water deficiency and salt stress, the WT plants all died, while the AtNHX5 overexpressing rice plants had a higher survival rate, dry weight, leaf water content, and leaf chlorophyll contents, accumulated more proline, and had less membrane damage than the WT plants. In addition, seeds of both transgenic and WT plants germinated on 1/2 MS medium supplemented with 250 mM mannitol, but overexpression of AtNHX5 improved the shoot growth of the seedlings. Taken together, the results indicate that AtNHX5 gene could enhance the tolerance of rice plants to multiple environmental stresses by promoting the accumulation of more effective osmolytes (ions or proline) to counter the osmotic stress caused by abiotic factors.     

国际水稻所水稻新株系的抗性评价

为了进一步扩大我国稻种资源,丰富水稻育种材料,引进了165份国际水稻研究所在非洲进行穿梭育种的水稻新株系,于2011年和2012年在湖北生态条件下进行稻瘟病抗性、白叶枯病抗性和褐飞虱抗性的评价。评价结果表明,在165份新株系中有14份株系在宜昌和恩施2个稻瘟病病圃鉴定均表现抗或中抗稻瘟病,有40份株系同时高抗或抗白叶枯病菌株ZHE173和GD1358,有19份株系抗或中抗褐飞虱,有7份株系同时抗白叶枯病和稻瘟病,有8份株系同时抗白叶枯病和褐飞虱,有1份株系同时中抗稻瘟病、褐飞虱和白叶枯病。部分材料正在作为中间材料用于水稻育种。     

过表达OsCatC基因水稻的获得及其耐盐性机理分析

过氧化氢酶C(Catalase C,CatC)作为重要的抗氧化酶,在水稻发育和胁迫响应方面起重要作用.为了探究CatC在盐胁迫响应中的功能及其作用机制,该研究构建了OsCatC过表达转基因水稻,并比较了其耐盐性和相关抗逆生理指标的变化.结果表明:(1)成功构建过量表达载体pCUbi1390-OsCatC-Flag,并经...     

Stability of sheath blight resistance in transgenic ASD16 rice lines expressing a rice <Emphasis Type="Italic">chi11</Emphasis> gene encoding chitinase

Development of transgenic plants by introducing defense genes is one of the strategies to engineer disease resistance. Transgenic ASD16 rice plants harbouring rice chitinase chi11 gene, belonging to a PR-3 group of defense gene conferring sheath blight (Rhizoctonia solani Kuhn) resistance, were used in this study. Three T₂ homozygous lines (ASD16-4-1-1, 5-1-1, and 6-1-1) were identified from seven putative (T₀) transgenic lines expressing chi11 using Western blotting analysis. The inheritance of sheath blight resistance in those lines was studied over generations. The stability of chi11 expression up to T₄ generation in all the three homozygous lines was proved by Western blot and the stability of sheath blight resistance in the homozygous lines was proved up to T₄ generation using detached leaf and intact leaf sheath assays. Among the three homozygous lines tested, ASD16-4-1-1 showed consistent results in all the generations and gave a better protection against the sheath blight pathogen than the other two lines.     

Overexpression of the wasabi defensin gene confers enhanced resistance to blast fungus ( Magnaporthe grisea) in transgenic rice

Transgenic rice ( Oryza sativa cv. Sasanishiki) overexpressing the wasabi defensin gene, a plant defensin effective against the rice blast fungus, was generated by Agrobacterium tumefaciens-mediated transformation. Twenty-two T2 homozygous lines harboring the wasabi defensin gene were challenged by the blast fungus. Transformants exhibited resistance to rice blast at various levels. The inheritance of the resistance over generations was investigated. T3 plants derived from two highly blast-resistant T2 lines (WT14-5 and WT43-5) were challenged with the blast fungus using the press-injured spots method. The average size of disease lesions of the transgenic line WT43-5 was reduced to about half of that of non-transgenic plants. The 5-kDa peptide, corresponding to the processed form of the wasabi defensin, was detected in the total protein fraction extracted from the T3 progeny. Transgenic rice plants overproducing wasabi defensin are expected to possess a durable and wide-spectrum resistance (i.e. field resistance) against various rice blast races.     

Overexpression of Rice Ferrochelatase I and II Leads to Increased Susceptibility to Oxyfluorfen Herbicide in Transgenic Rice

Protoporphyrin IX is a photosensitizer and a causative agent of rice membrane lipid peroxidation in plant cells. Protoporphyrinogen IX oxidase (PPO) is the molecular target of PPO-inhibiting herbicides, which trigger a massive increase in protoporphyrin IX. Thus, any possible method to decrease the levels of protoporphyrin IX upon challenge with PPO-inhibiting herbicides could be employed to generate plants resistant to such herbicides. We generated transgenic rice plants overexpressing rice ferrochelatase isogenes encoding ferrochelatase enzymes, which convert protoporphyrin IX into protoheme, to see whether the transgenic plants have phenotypes resistant to PPO-inhibiting herbicides. The resulting transgenic rice plants were all susceptible to oxyfluorfen (a diphenyl-ether-type PPO-inhibiting herbicide), as judged by cellular damage with respect to cellular leakage, chlorophyll loss, and lipid peroxidation. In particular, the transgenic plants expressing rice ferrochelatase II without its plastid targeting sequence showed higher transgene expression and oxyfluorfen susceptibility than lines expressing the intact ferrochelatase II. Possible susceptibility mechanisms to oxyfluorfen herbicide in the transgenic rice plants are discussed.