Rice blast is one of the most serious diseases of rice and a major threat to rice production. Breeding disease-resistant rice is one of the most economical, safe, and effective measures for the control of rice blast. As a complement to traditional crop breeding, the transgenic method can avoid the time-consuming process of crosses and multi-generation selection. In this study, maize (Zea mays) Activator (Ac)/Dissociation (Ds) transposon vectors carrying green fluorescent protein (GFP) and red fluorescent protein (mCherry) genetic markers were used for generating marker-free transgenic rice. Double fluorescent protein-aided counterselection against the presence of T-DNA was performed together with polymerase chain reaction (PCR)-based positive selection for the gene of interest (GOI) to screen marker-free progeny. We cloned an RNAi expression cassette of the rice Pi21 gene that negatively regulates resistance to rice blast as a GOI into the Ds element in the Ac/Ds vector and obtained marker-free T1 rice plants from 13 independent transgenic lines. Marker-free and Ds/GOI-homozygous rice lines were verified by PCR and Southern hybridization analysis to be completely free of transgenic markers and T-DNA sequences. qRT-PCR analysis and rice blast disease inoculation confirmed that the marker-free transgenic rice lines exhibited decreased Pi21 expression levels and increased resistance to rice blast. TAIL-PCR results showed that the Ds (Pi21-RNAi) transgenes in two rice lines were reintegrated in intergenic regions in the rice genome. The Ac/Ds vector with dual fluorescent protein markers offers more reliable screening of marker-free transgenic progeny and can be utilized in the transgenic breeding of rice disease resistance and other agronomic traits.

[目的]在转录水平上解析Pi9基因介导的稻瘟病抗性调控机理,为培育抗病水稻品种提供理论依据.[方法]向水稻品种日本晴(NPB)及其转Pi9抗稻瘟病基因株系(NPB/Pi9)接种稻瘟菌.分别于接种后0 h、12 h、24 h、36 h提取叶组织样品,选取12503个水稻基因定制基因芯片,进行水稻基因转录组分析,并通过qRT-PCR对部分差异表达基因进行验证.[结果]NPB/Pi9在接种后12 h、24 h和36 h的基因表达量分别与其接种0 h表达量比较,共检测到7754个差异表达基因;相应地,感病水稻NPB在以上时间点共检测到7385个差异表达基因;在接种后36 h,NPB/Pi9的差异表达基因数目显著多于NPB.比较NPB/Pi9和NPB相同时间点的基因表达量,共获得4065个差异表达基因,其中接种后36 h的差异表达基因显著多于接种后0 h、12 h或24 h.因此,NPB/Pi9的稻瘟病防御反应更强烈.对NPB/Pi9与NPB相同时间点的差异表达基因进行GO和KEGG分析,细胞外区域、植物对刺激应答、转录调控、氧化还原、离子结合、次生代谢和植物激素相关的GO分类在接种后呈显著富集,苯丙氨酸代谢、类黄酮生物合成和植物激素信号途径的KEGG通路在接种后显著富集.与效应分子触发的免疫反应(ETI)相关的水杨酸信号途径、几丁质酶,以及与病原相关分子模式触发的免疫反应(PTI)相关的胞外区域、对刺激的应答、木质素合成等,均在抗感水稻之间差异表达.而且PTI/ETI共有的WRKY转录因子、MAPK激酶、茉莉酸和乙烯信号途径等发生差异表达.综上所述,NPB/Pi9和NPB的差异表达模式与ETI和PTI相关,两者相互联系并在Pi9介导的稻瘟病抗性中发挥作用.[结论]与日本晴比较,抗病基因型NPB/Pi9对稻瘟病防御反应更强烈.转录因子、激酶、NBS-LRR基因、几丁质酶、水杨酸、茉莉酸和乙烯信号途径,以及植物次生代谢在Pi9介导的稻瘟病抗病反应中发挥重要作用.

Rice sheath blight (SB), caused by Rhizoctonia solani ( R. solani ), is a major threat to rice production worldwide. The molecular mechanisms of the SB resistance in rice are poorly understood. The transcriptomes of the SBresistant rice cultivar YSBR1 and the susceptible cultivar Lemont were analyzed after R. solani infection. A total of 7624 differentially expressed genes (DEGs) were identified at one or more timepoints in a cultivar. 5526 and 5618 DEGs were differentially expressed in Lemont and YSBR1, respectively. YSBR1 exhibited stronger and earlier transcriptional response to R. solani than Lemont. Gene ontology enrichment analysis revealed that genes that encode cell wall-modifying and glycosyl-degrading enzymes or anti-microbial proteins were specifically induced in YSBR1 at 6 hpi. MapMan analysis revealed that more DEGs related with cell wall, β-glucanses, respiratory burst, phenylpropanoids and lignin were highly induced by R. solani in YSBR1 than in Lemont. The results also showed that receptor-like kinases and jasmonic acid signaling may play important roles in host resistance to R. solani . This study highlights potential candidate genes and signaling pathways involved in rice sheath resistance and can help to further clarify the mechanistic events underlying resistance and susceptibility to R. solani .

pi21 gene was targeted editing using CRISPR/Cas9 technology in the blast susceptible rice variety Nipponbare and a set ofpi21 recessive mutants exhibiting enhanced rice blast resistance were obtained.The results showed that 86.7％ of T0 transgenic plants had nucleotide deletions ofpi21 target sequence.Through T-DNA specific PCR,the T1 generation plants containing T-DNA were eliminated,and 107 T-DNA-free plants were identified from 23 T1 transgenic lines.Restriction digestion and sequencing analysis revealed that 21 out of 107 rice plants were pi21 homozygous mutants.One pi21 mutant line was inoculated with M.oryzae,showing significantly enhanced rice blast resistance compared with the untransformed Nipponbare.qRT-PCR analysis showed that the expression of the tested pathogenesis-related genes were more rapidly and strongly induced in the pi21 mutant plants compared to the blast susceptible Nipponbare during fungal infection.In this study,the targeted editing ofpi21 gene in the blast susceptible variety and improved resistance to rice blast was achieved,which provides the foundation for breeding durable rice blast resistant rice varieties.

过量表达是植物基因功能鉴定和农作物遗传改良的重要途径.为了提高转基因植株的研究效率,以mCherry红色荧光蛋白为报告基因,构建了由玉米泛素启动子(P-Ubi)和豌豆T3A-polyA序列组成过量表达框的转基因载体.分别将4个水稻类受体激酶的编码序列插入表达框,通过农杆菌介导转化水稻品种泰粳394.转基因植株T1种子的mCherry荧光检测结果表明,平均62.5％的T1株系呈3:1分离.根据实时定量RT-PCR分析结果,过表达阳性植株的目的基因相对表达量显著高于阴性植株和未转化的泰粳394.运用该载体系统,能够进行转基因后代大规模筛选,获得目的基因过量表达的转基因株系,从而促进植物基因功能研究和转基因应用研究.

Marker-free transgenic plants can be developed through transposon-mediated transgene reintegration, which allows intact transgene insertion with defined boundaries and requires only a few primary transformants. In this study, we improved the selection strategy and validated that the maize (Zea mays) Activator/Dissociation (Ds) transposable element can be routinely used to generate marker-free transgenic plants. A Ds-based gene of interest was linked to green fluorescent protein in transfer DNA (T-DNA), and a green fluorescent protein-aided counterselection against T-DNA was used together with polymerase chain reaction (PCR)-based positive selection for the gene of interest to screen marker-free progeny. To test the efficacy of this strategy, we cloned the Bacillus thuringiensis (Bt) δ-endotoxin gene into the Ds elements and transformed transposon vectors into rice (Oryza sativa) cultivars via Agrobacterium tumefaciens. PCR assays of the transposon empty donor site exhibited transposition in somatic cells in 60.5% to 100% of the rice transformants. Marker-free (T-DNA-free) transgenic rice plants derived from unlinked germinal transposition were obtained from the T1 generation of 26.1% of the primary transformants. Individual marker-free transgenic rice lines were subjected to thermal asymmetric interlaced-PCR to determine Ds(Bt) reintegration positions, reverse transcription-PCR and enzyme-linked immunosorbent assay to detect Bt expression levels, and bioassays to confirm resistance against the striped stem borer Chilo suppressalis. Overall, we efficiently generated marker-free transgenic plants with optimized transgene insertion and expression. The transposon-mediated marker-free platform established in this study can be used in rice and possibly in other important crops.

State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control and Institute of Virology and Biotechnology (X.G., J.Zho., J.L., X.Z., J.Zha., Y.T., S.Q.), Institute of Crop Science and Nuclear Technology Utilization (D.W.), and Institute of Quality Standards for Agricultural Products (X.C.), Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China; Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (Q.L., R.X., Q.X.); and Institute of Crop Science (R.Y., J.T.) and Institute of Insect Sciences (Z.Zu., Z.Zh.), Zhejiang University, Hangzhou, Zhejiang 310058, China