Elevating plant immunity by translational regulation of a rice WRKY transcription factor.

Plants have intricate mechanisms that tailor their defence responses to pathogens. WRKY transcription factors play a pivotal role in plant immunity by regulating various defence signalling pathways. Many WRKY genes are transcriptionally activated upon pathogen attack, but how their functions are regulated after transcription remains elusive. Here, we show that OsWRKY7 functions as a crucial positive regulator of rice basal immunity against Xanthomonas oryzae pv. oryzae (Xoo). The activity of OsWRKY7 was regulated at both translational and post-translational levels. Two translational products of OsWRKY7 were generated by alternative initiation. The full-length OsWRKY7 protein is normally degraded by the ubiquitin-proteasome system but was accumulated following elicitor or pathogen treatment, whereas the alternate product initiated from the downstream in-frame start codon was stable. Both the full and alternate OsWRKY7 proteins have transcriptional activities in yeast and rice cells, and overexpression of each form enhanced resistance to Xoo infection. Furthermore, disruption of the main AUG in rice increased the endogenous translation of the alternate stabilized form of OsWRKY7 and enhanced bacterial blight resistance. This study provides insights into the coordination of alternative translation and protein stability in the regulation of plant growth and basal defence mediated by the OsWRKY7 transcription factor, and also suggests a promising strategy to breed disease-resistant rice by translation initiation control.

Ferredoxin 1 is downregulated by the accumulation of abscisic acid in an ABI5-dependent manner to facilitate rice stripe virus infection in Nicotiana benthamiana and rice.

Ferredoxin 1 (FD1) accepts and distributes electrons in the electron transfer chain of plants. Its expression is universally downregulated by viruses and its roles in plant immunity have been brought into focus over the past decade. However, the mechanism by which viruses regulate FD1 remains to be defined. In a previous report, we found that the expression of Nicotiana benthamiana FD1 (NbFD1) was downregulated following infection with potato virus X (PVX) and that NbFD1 regulates callose deposition at plasmodesmata to play a role in defense against PVX infection. We now report that NbFD1 is downregulated by rice stripe virus (RSV) infection and that silencing of NbFD1 also facilitates RSV infection, while viral infection was inhibited in a transgenic line overexpressing NbFD1, indicating that NbFD1 also functions in defense against RSV infection. Next, a RSV-derived small interfering RNA was identified that contributes to the downregulation of FD1 transcripts. Further analysis showed that the abscisic acid (ABA) which accumulates in RSV-infected plants also represses NbFD1 transcription. It does this by stimulating expression of ABA insensitive 5 (ABI5), which binds the ABA response element motifs in the NbFD1 promoter, resulting in negative regulation. Regulation of FD1 by ABA was also confirmed in RSV-infected plants of the natural host rice. The results therefore suggest a mechanism by which virus regulates chloroplast-related genes to suppress their defense roles.

OsTGAL1 suppresses the resistance of rice to bacterial blight disease by regulating the expression of salicylic acid glucosyltransferase OsSGT1.

Many TGA transcription factors participate in immune responses in the SA-mediated signaling pathway in Arabidopsis. This study identified a transcription factor OsTGAL1, which is induced upon infection by Xoo. Overexpression of OsTGAL1 increased the susceptibility of rice to Xoo. Plants overexpressing OsTGAL1 could affect the expression of many SA signaling-related genes. OsTGAL1 was able to interact with the promoter of OsSGT1, which encodes a key enzyme for SA metabolism. The transcript of OsSGT1 was induced by Xoo and this responsive expression was further increased in plants overexpressing OsTGAL1. OsSGT1 knockout lines had enhanced resistance to Xoo, and knocking out OsSGT1 in plants overexpressing OsTGAL1 blocked the susceptibility caused by OsTGAL1. Altered expression levels of several OsPRs in all the transgenic plants demonstrated that SA-mediated signaling had been affected. Furthermore, we identified an oxidoreductase of CC-type glutaredoxin, OsGRX17, which interacted with OsTGAL1. OsGRX17 reduced the regulation of OsTGAL1 on OsSGT1, and this may be due to its redox modulation. Thus, our results demonstrate that OsTGAL1 negatively regulates resistance to Xoo by its effects on SA metabolism via the activation of OsSGT1, which provides valuable targets for plant breeders in developing new cultivars that are resistant to Xoo.

Targeted Transgene Expression in Rice Using a Callus Strong Promoter for Selectable Marker Gene Control.

Precise expression of a transgene in the desired manner is important for plant genetic engineering and gene function deciphering, but it is a challenge to obtain specific transgene expression free from the interference of the constitutive promoters used to express the selectable marker gene, such as the Cauliflower mosaic virus (CaMV) 35S promoter. So, the solutions to avoid these inappropriate regulations are largely demanded. In this study, we report the characterization of a callus strong promoter (CSP1) in rice and its application for accurate transgene expression. Our results indicate that the high expression of the CSP1 promoter in the callus enables efficient selection of hygromycin equivalent to that provided by the CaMV 35S promoter, whereas its expression in other tissues is low. To evaluate possible leaky effects, the expression of a ß-glucuronidase reporter driven by six specific promoters involving hormone signaling, pathogen response, cell fate determination, and proliferation was observed in transgenic rice plants generated by CSP1-mediated selection. Distinct ß-glucuronidase expression was found consistently in most of the transgenic lines obtained for each promoter. In addition, we applied these specific marker lines to investigate the root cellular responses to exogenous cytokinin and auxin treatment. The results reveal that the root growth inhibition by cytokinin was differently regulated at high and low concentrations. In summary, we have established the feasibility of using callus-specific promoter-dependent selection to mitigate the transgene misexpression in rice. By enabling efficient transformation, rice plants with reliable transgene expression will be easily acquired for broad applications.

pGP-B2E, a Recombinant Compatible TA/TB-Ligation Vector for Rapid and Inexpensive Gene Cloning.

DNA cloning is the basic step for different fields of life science, and many efforts have been made to simplify this procedure. In this study, we report two general purpose plasmids (pGP), pGP-XB2E and pGP-B2E, for rapid and cost-effective construct of basic clones. The BciVI and XcmI cleavage sites are designed in pGP-XB2E to test plasmid linearization efficiency. The plasmid has better linearization efficiency by using BciVI which could almost completely digest 2 µg plasmid in 10 min with only one-tenth the recommended enzyme concentration. In order to further optimize the pGP-XB2E, a new plasmid, pGP-B2E, which removed XcmI cleavage site was designed. This vector is highly efficient for cloning PCR products up to 1812 bp, and the number of colonies was about five times that of pGP-XB2E. In addition to TA cloning, blunt-end PCR products with T ended in the primer could be positively linked to the T-vector pGP-B2E without A-tailing treatment (TB cloning). Moreover, as an entry vector, pGP-B2E was also compatible for gateway recombination reaction without frameshift mutations. In general, this vector provides a universal, quick, and cost-efficient method for basic molecular cloning.