| Rice (Oryza sativa L) is one of the most important food crops in China. Rice blast, caused by the fungal pathogen Magnaporthe oiyzae, is one of most devastated diseases in rice production, and is responsible for annual 15%-20% yield loss, even up to 40%-50% or nothing to harvest in epidemic years. Therefore, prevention and control of rice blast is very important for rice production and food security. Heat shock protein (HSP) is a large family that can be divided into five evolutionarily conserved groups according to molecular mass: small HSPs (sHSPs), HSP60, HSP70, HSP90 and HSP100. Hsp40 is a kind of sHSPs with the molecular weight of 40 kDa. It has been reported that Hsp40 participates in the antiviral defense pathway in plants such as Nicotiana benthamiana and rice, but its function in response to fungal pathogens is not clear. In this study, we selected one upregulated gene from the MPSS (Massively Parallel Signature Sequencing) data of rice inoculated with M. oiyzae. We made RNAi constructs of this gene and generated transgenic lines. This rice DnaJ gene OsDjA6 was identified from the phenotype evaluation of the genetically modified (gm) homozygou lines. The functional analysis results of OsDjA6 are summarized as follows:1.MPSS data showed that, after 12 h inoculation of compatible and incompatible isolates of M. oryzae, OsDjA6 was induced in the incompatible reaction, but not in the compatible and the control. These results suggest that this gene might be involved in defense response against M. oryzae;2. We found that this gene encoded a J-domain containing protein through sequence analysis and alignment, which bolongs to the Type A DnaJ protein and it was named OsDjA6;3. By using Agrobacteriwn-mediated transformation method, the RNAi vectors were transformed into japonica rice cultivar TG394, and transgenic lines were generated. Two methods including hygromycin resistance gene and mCherry red fluorescence detection were used to identify the positive transgenic plants. qRT-PCR analysis confirmed a significant reduction of OsDjA6 gene expression in the positive RNAi transgenic lines;4. T2 gm homozygou seedlings were used for spraying inoculation with M. oryzae isolate RO1-1. The inoculation experiment was repeated for three times, and then three independent RNAi lines were chosen for "punch" inoculation. The inoculations showed that the RNAi lines of OsDjA6 displayed significantly enhanced resistance to RO1-1;5. We examined the response of the TG394 and OsD/A6 RNAi plants to the PAMPelicitors flg22 and chitin. In flg22-treated tissues, silencing of OsDjA6 resulted in an increase in ROS production compared with the wild-type TG394. While when treated with chitin, there were no significant difference. This indicates that OsDjA6 may play its role in rice PTI (pattern triggered immunity);6. Fluorescence quantitative PCR detected up-regulated expression of three defense-related genes PR5, NH1 and WRKY45, suggesting that OsDjA6 could be involved in defense response against rice blast through regulating the SA signal pathway;7. Transient expression assays through rice protoplasts system demonstrated that OsDjA6 was localized in both the cell membrane and nucleus, and deletion of different domains including J-domain, ZF-domain of OsDjA6 did not affect its position in the cell;8. Using BD-OsDjA6 as a bait to screen rice cDNA libraries, we identified an C3HC4 E3 ligase to interact with OsDjA6 in yeast. In addition, OsSGTl, a key regulator of rice resistant to M. oryzae, could be suppressed by OsDjA6 in yeast.These results lay a foundation for further study on the function of OsDjA6 in the interaction with M. oryzae and also provide new strategy to engineer resistant rice plants by silencing HSP genes. |
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