【Objective】 Rice blast is one of the main diseases that threaten rice yield and quality. OsBSK1-2 has been found to be involved in rice blast resistance regulation. Previously, OsCBSX4, a cysteine sulfide-β-Synthase, was identified by screening the proteins that interact with OsBSK1-2. The purpose of this study was to verify the interaction between OsBSK1-2 and OsCBSX4, and clarify the function and molecular mechanism of OsCBSX4 in rice blast resistance, providing a theoretical basis for rice disease resistance breeding.【Method】 Co-immunoprecipitation, bimolecular fluorescence complementation and luciferase complementation assays were used to determine the interaction between OsBSK1-2 and OsCBSX4. Then, quantitative PCR and agrobacterium- mediated transient transformation in N. benthamiana were used to detect the gene expression pattern and protein localization of OsCBSX4. Subsequently, OsCBSX4-knockingout and OsCBSX4-overexpressing plants were generated via CRISPR/Cas9 technology and Agrobacterium-mediated genetic transformation, respectively, and their resistance to rice blast was determined by inoculation with M. oryzae. Moreover, the immune responses induced by chitin and M. oryzae in the oscbsx4 mutant were analyzed using ROS burst and DAB staining assays respectively. In the end, the interaction between OsCBSX4 and OsRbohB was verified via bimolecular fluorescence complementation and split-luciferase complementation imaging assays, and the impact of the metabolite of OsCBSX4 on rice blast resistance was determined using the detached leaf inoculation method. The studies above will reveal the immune function and molecular mechanism of OsCBSX4.【Result】 The interaction between OsCBSX4 and OsBSK1-2 was verified by CoIP, LCI and BiFC assays. Compared to the wild type, the oscbsx4 mutant showed more disease lesions after inoculation with M. oryzae by the spray method, as well as a larger lesion area and greater fungal growth after inoculation with M. oryzae by the punch method, suggesting that knocking out OsCBSX4 decreased rice blast resistance. Moreover, the expression of the pathogenesis-related genes, OsPR5 and OsPR10, and the H2O2 accumulation induced by M. oryzae infection and the ROS burst induced by chitin treatment were reduced in the oscbsx4 mutant. Compared with the wild type, OsCBSX4 overexpressing plants showed a smaller lesion area and less fungal growth after inoculation with M. oryzae by the punch method, suggesting that overexpression of OsCBSX4 increased rice blast resistance. In addition, we found that OsCBSX4 can interact with OsRbohB, a key regulator of rice ROS production and treating rice with L-cysteine, a metabolite of OsCBSX4, does not affect rice blast resistance. 【Conclusion】 OsCBSX4 is an important component of OsBSK1-2 signaling and positively regulates rice blast resistance. OsCBSX4 may mediate ROS production by interacting with OsRbohB, thereby regulating rice immunity.
【Objective】 Acuce, currently the predominant cultivating rice landrace at Yuanyang Hani terrace, Yunnan Province, has been continuously planted for more than 100 years. Interestingly, there has never been a major outbreak of rice blast during the past decades. Clarifying the types, quantities, expression characteristics, and phenotypic effects of functional genes involved in the resistance response process so as to exploring the molecular mechanism of Acuce resistance to Magnaporthe oryzae will optimize facilitate the conservation and utilization of rice landrace. 【Method】 The present research focuses on the biological effect of disease resistance-related candidate gene BGIOSGA023826 which screened from Acuce -M. oryzae interaction transcriptome. Initially, biological function of this gene was analyzed by using bioinformatics software and conducted gene cloning. Subsequently, overexpression vector was constructed through Agrobacterium-mediated genetic transformation, then carry out positive identification and disease resistance phenotype analysis of overexpressed plants. Finally, Real-time fluorescence quantitative PCR was used to analyze the expression of PR genes in overexpressed plants, and enzyme-linked immunosorbent assay was used to determine changes in endogenous hormone content. 【Result】 The candidate gene BGIOSGA023826 encodes 354 amino acids figuring unstable hydrophobic characteristic without transmembrane domains, coding a non-membrane and non-secretory protein, classified as a hydrolase gene. The overexpression of BGIOSGA023826 in transgenic plants significantly enhances resistance to M. oryzae compared to Nipponbare. The results of real-time fluorescent quantitative PCR indicate that the relative expression levels of the PR genes PR1b, OsNPR1, and OsAOS2 as well as transcription factors WRKY45 significantly upregulated. Both of the PR1b gene and the key gene OsAOS2 functioning in the JA synthesis pathway continuously upregulated from 12 hpi. Meanwhile, the expression level of the transcription factor gene WRKY45 drastically increased at 24 hpi and reached its peak at 36 hpi, which were nearly two folds higher than that of Nipponbare. As to the results of endogenous hormone content measurement, the JA increased to 263.88 pmol·L-1 at 12 hpi, reaching peak 304 pmol·L-1 at 36 hpi; Similarly, the SA increased to 186.5 pmol·L-1 at 24 hpi, reaching peak 198.88 pmol·L-1 at 36 hpi; At the same time, the content of ROS also rises continually and reached its peak at 36 hpi, MDA also increased from 40.43 nmol·g-1 to 53.94 nmol·g-1; However, SOD did not exhibit significant differences at individual time points in transgenic plant, but showed an upward trend in Nipponbare. 【Conclusion】 Preliminary elucidating of the temporal expression relationship between the hydrolase gene BGIOSGA023826 and the PR genes as well as endogenous hormone levels, demonstrating it involved in the landrace Acuce resistance to M. oryzae infection process.
【Objective】 This study aims to discover new quantitative trait loci (QTLs) for resistance to the brown planthopper (BPH) in rice by utilizing a genetic population and to assess the pyramiding effects of these QTLs, thereby providing crucial genetic materials and resources for breeding insect-resistant rice. 【Method】 A recombinant inbred line (RIL) population derived from a cross between susceptible NPB and resistant Jiafuzhan was used, combined with seedling stage resistance evaluation and extreme mixed pool sequencing (BSA-seq) to map BPH resistance QTLs. Further, fine mapping, candidate gene identification, pyramiding effect analysis, and mixed-pool transcriptome sequencing (BSR-seq) were employed to elucidate the physiological and molecular mechanisms mediated by these QTLs. 【Result】 BSA-seq identified two major resistance QTLs on chromosomes 1 (30-32 Mb) and 4 (5-7 Mb), named QBPH1 and QBPH4, respectively. Interval-linked molecular markers confirmed the authenticity of these QTLs. QBPH4 coincides with previously cloned BPH3 and BPH15, while QBPH1 represents a newly discovered QTL. High-density markers and recombinant analysis further narrowed the QBPH1 region to 30.61-30.65 Mb. This analysis identified Os01g53294 and Os01g53330 as reliable candidate genes, which code for a respiratory burst oxidase protein B and an anthocyanin 5,3-O-glucosyltransferase, respectively. In evaluations of seedling resistance, brown planthopper honeydew area and weight, insect weight gain, lethality, and preference, comparisons between QBPH1 and QBPH4 single genes and their polymerized lines revealed no significant enhancement in resistance. Both QBPH1 and QBPH4 mediated antibiosis and antixenosis mechanisms with varying effects. BSR-seq analysis highlighted significant enrichment of differentially expressed genes (DEGs) involved in transcriptional regulation, protein phosphorylation, and redox processes among different QBPH1 alleles. Additionally, genes associated with jasmonic acid (JA) synthesis and signaling pathways were significantly upregulated in resistant materials, confirmed by RT-qPCR experiments. 【Conclusion】 A novel BPH resistance QTL, QBPH1, was successfully identified on chromosome 1 of rice. QBPH1 mediated both antibiosis and avoidance against BPH, though its aggregation effect with another QTL was not significant. QBPH1 may mediate defense mechanisms against BPH through involvement in the JA pathway. Based on this, Os01g53294 and Os01g53330 have been validated as reliable candidate genes for QBPH1.
【Objective】 Soybean mosaic virus (SMV) disease causes by SMV seriously restricts soybean yields and qualities. In this study, we cloned GmRHF1, which encodes a Ring-H2 type zinc finger protein (RHF), and identified its function in SMV resistance through the sequence variation analysis and the virus induced gene silencing (VIGS) assay. Yeast two-hybrid and luciferase complementation assay (LCA) were used to screen and verify the interacting proteins of GmRHF1. The research lay a foundation for further exploring the mechanism of GmRHF1 in soybean resistance to SMV.【Method】 Firstly, GmRHF1 was cloned from soybean varieties of Xudou 14 (highly resistant to SMV) and Shishanheidou (highly susceptible to SMV), and the consistency of sequence variation was then analyzed in representative resistant and susceptible soybean resources, so as to excavate the excellent alleles with SMV resistance. Secondly, the expression abundance of GmRHF1 in different soybean tissues and under SMV treatment were analyzed by real-time quantitative PCR (RT-qPCR), respectively. At the same time, the function of GmRHF1 in SMV resistance was identified by the VIGS assay. The interacting proteins of GmRHF1 were screened and verified by yeast cDNA library screening, point to point verification and LCA experiments. Finally, the function of GmClpP6 on SMV resistance was verified, and the biological significance based on the interaction of GmRHF1 and GmClpP6 for soybean resistance to SMV was clarified.【Result】 GmRHF1 contained a conserved RING-H2 domain of the E3 ubiquitin protein ligase, and belongs to the typical RING-H2 finger family. The results of sequence variation analysis showed that a natural non-synonymous mutation existed in the CDS region of GmRHF1 among various soybean varieties, providing a probable relevance to the SMV resistance or sensibility. After SMV inoculations, the expression level of GmRHF1 in resistant soybean was significantly higher than that in susceptible material by RT-qPCR assay. Through the VIGS experiments, GmRHF1 was effectively silenced and the soybean leaves showed a weakened SMV resistance than the normal plants, indicating that GmRHF1 could enhance soybean resistance to SMV. Based on the yeast cDNA library, 30 potential interacting proteins of GmRHF1 were screened. The interaction between GmRHF1 and GmClpP6 was confirmed by the two-hybrid and LCA experiments. Furthermore, the soybean resistance to SMV was also reduced in soybean leaves with the silenced GmClpP6 by the VIGS experiments.【Conclusion】 The non-synonymous mutation identified in the coding sequence of GmRHF1 may be a key SNP variation related to the SMV resistance and sensibility; and GmRHF1 can interact with GmClpP6. GmRHF1 plays an important role in SMV resistance.
