Data_Sheet_1_Heat Shock Protein HSP24 Is Involved in the BABA-Induced Resistance to Fungal Pathogen in Postharvest Grapes Underlying an NPR1-Dependent Manner.doc

Although heat shock proteins (HSPs), a family of ubiquitous molecular chaperones, are well characterized in heat stress-related responses, their function in plant defense remains largely unclear. Here, we report the role of VvHSP24, a class B HSP from Vitis vinifera, in β-aminobutyric acid (BABA)-induced priming defense against the necrotrophic fungus Botrytis cinerea in grapes. Grapes treated with 10 mmol L–1 BABA exhibited transiently increased transcript levels of VvNPR1 and several SA-inducible genes, including PR1, PR2, and PR5. Additionally, phytoalexins accumulated upon inoculation with the gray mold fungus B. cinerea, which coincided with the action of a priming mode implicated in pathogen-driven resistance. Intriguingly, electrophoretic mobility shift (EMSA), yeast two-hybrid (Y2H) and His pull-down assays demonstrated that the nuclear chaperone VvHSP24 cannot modulate the transcript of PR genes but does directly interact with VvNPR1 in vivo or in vitro. Furthermore, we found that VvHSP24 overexpression enhanced the transcript levels of NPR1 and SA-responsive genes (PR1, PR2, and PR5) and increased the resistance of transgenic Arabidopsis thaliana to B. cinerea compared with wildtype Col-0. An opposite trend between CRISPR mutants of AtHSFB1 (the orthologous gene of VvHSP24 in Arabidopsis) and wildtype plants was observed. Hence, our results suggest that VvHSP24 has a potential role in NPR1-dependent plant resistance to fungal pathogen. BABA-induced priming defense in grapes may require posttranslational modification of the chaperone VvHSP24 to activate VvNPR1 transcript, leading to PR gene expressions and resistance phenotypes.

热休克蛋白（heat shock proteins, HSPs）是一类广泛分布的分子伴侣（molecular chaperones）家族，尽管其在热胁迫相关应答中的功能已得到充分解析，但它们在植物防御中的作用仍未被明确阐明。本研究报道了葡萄（Vitis vinifera）来源的B类热休克蛋白VvHSP24在β-氨基丁酸（β-aminobutyric acid, BABA）诱导葡萄对抗死体营养型真菌灰葡萄孢（Botrytis cinerea）的启动防御中的作用。经10 mmol·L⁻¹ BABA处理的葡萄，其VvNPR1以及PR1、PR2、PR5等水杨酸诱导基因的转录水平呈现瞬时上调。此外，接种灰霉菌B. cinerea后，葡萄体内植保素显著积累，这与病原体驱动的抗性相关的启动防御模式的作用相一致。有趣的是，电泳迁移率变动分析（electrophoretic mobility shift assay, EMSA）、酵母双杂交（yeast two-hybrid, Y2H）以及组氨酸标签蛋白下拉实验（His pull-down）结果显示，核分子伴侣VvHSP24无法调控病程相关蛋白（PR）基因的转录，但可在体内外直接与VvNPR1相互作用。进一步研究发现，与野生型Col-0相比，VvHSP24过表达可上调拟南芥（Arabidopsis thaliana）中NPR1以及水杨酸响应基因（PR1、PR2、PR5）的转录水平，并增强转基因拟南芥对灰葡萄孢的抗性。而拟南芥中VvHSP24的直系同源基因AtHSFB1的CRISPR突变体，则呈现出相反的表型趋势。综上，本研究结果表明，VvHSP24在依赖于NPR1的植物对抗真菌病原体的抗性中具有潜在作用。葡萄体内BABA诱导的启动防御，可能需要通过对分子伴侣VvHSP24进行翻译后修饰，以激活VvNPR1的转录，进而引发PR基因的表达与抗性表型的形成。