由果生刺盘孢引起的炭疽叶枯病是我国苹果产区的重要病害。果生刺盘孢在侵染苹果过程中分泌效应蛋白CfEC92促进其侵染，但其作用机制仍不清楚。本研究从苹果cDNA中克隆出Mal d 1j蛋白，结构域及氨基酸序列分析显示，Mal d 1j为PR10家族成员。
Mal d 1j基因过表达能显著增 强苹果对炭疽叶枯病抗性，而沉默Mal d 1j显著降低其抗性。在*草烟**中过表达Mal d 1j提高*草烟**抗 性，共表达CfEC92和Mal d 1j蛋白，降低Mal d 1j对疫霉菌抗性。
苹果
通过酵母双杂交、BIFC和Co-IP分析证明CfEC92与Mal d 1j可以发生直接互作，且其互作定位于细胞膜和细胞核，表明CfEC92通过与Mal d 1j互作影响植物免疫。本研究揭示了果生刺盘孢效应蛋白CfEC92通过靶向Mal d 1j 抑制植物免疫促进其侵染的分子机制，为苹果炭疽叶枯病防控提供了新的思路。
供试菌株
实验所用果生刺盘孢Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde 野生型1104-7与*草烟**疫霉Phytophthora nicotianae Breda de Haan由西北农林科技大学植物保护学院苹果真菌病害研究室保存。
Mal d 1j系统发育分析及同源序列比对
从Uniprot网站搜寻Mal d 1j同源蛋白以及已知功能PR10蛋白，*载下**氨基酸序列。用 MEGA7.0软件对靶标氨基酸序列进行系统发育分析(neighbor-joining 法)。用 DANMAN软件进行序列比对。
农杆菌介导的过表达和沉默
苹果果实过表达Mal d 1j：将转入pBin-Mal d 1j的GV3101农杆菌菌液注射苹果果实，注射pBin空载为对照。将注射过的苹果果实黑暗放置30h，采用qRT-PCR检测基因表达量。将果生刺盘孢野生型菌饼接种于注射孔附近，用脱脂棉进行保湿，在培养箱中黑暗保育4d (28 ℃)， 观察其发病情况。
沉默苹果果实Mal d 1j：将TRV2、TRV2-Mal d 1j分别与TRV1菌液1:1混匀，注射苹果果实， 将注射过的苹果果实黑暗下喷水保育15d，用qRT-PCR检测基因表达量，其他处理同过表达 处理。
苹果
*草烟**叶片过表达Mal d 1j：将转入1300-Mal d 1j 的农杆菌菌液注射本氏*草烟**，注射1300空载的农杆菌菌液作为对照，2d后观察荧光，运用qRT-PCR技术检测基因表达量，对过表达的 *草烟**接种*草烟**疫霉，24h之后观察发病情况。
*草烟**叶片
*草烟**叶片共表达CfEC92与Mal d 1j：将转入1300-Mal d 1j与pBin-CfEC92的农杆菌菌液1:1 混匀，注射本氏*草烟**，注射1300-Mal d 1j的农杆菌作为对照，其他处理同过表达处理。
酵母双杂交互作验证
从苹果cDNA 中扩增Mal d 1j，构建pGADT7-Mal d 1j载体，利用酵母共转化的方法将pGBKT7-CfEC92与pGADT7-Mal d 1j共同转入酵母菌株 Y2HGold，其菌液涂布于缺陷型培养基DDO (SD/-Trp/-Leu)，30℃培养3d，挑取菌落摇菌后，滴在缺陷性培养基TDO/X/A (SD/-Trp/-Leu/-His，X-α-gal，aba)上，30℃培养3–5d，挑取蓝色菌落摇菌，滴在QDO/X/A (SD/- Trp/-Leu/-His/-Ade，X-α-gal，aba)上，30℃培养5d，观察菌落形态及颜色。
双分子荧光互补互作验证
以Blunt Zero-CfEC92和pGADT7-Mal d 1j质粒为模板，扩增CfEC92和Mal d 1j片段，分别插入YFPn 和YFPc载体中，获得YFPn-CfEC92 和YFPc-Mal d 1j质粒，将载体质粒转入农杆菌感受态GV3101中，挑取正确菌落进行摇培，将菌液收集并用MgCl2重悬液(每 200mL含MgCl2 0.4g，MES 0.4g，100mmol/L乙酰丁香酮200μL) 调吸光度值为0.6时，共注射本氏*草烟**，36–48h观察YFP荧光。
1.6Co-IP互作验证构建pBin-CfEC92-GFP 与1300-mCherry-Mald 1j 载体，转入农杆菌感受态GV3101中，注射本氏*草烟**，方法同1.5。
48–72h 观察GFP 与 mCherry荧光共定位。收集样品，研磨并加入RIP提取液(1 mmol/L DTT，1mmol/L蛋白酶*制剂抑**，1mmol/L PMSF)提取*草烟**总蛋白，以GFP抗体磁珠富集蛋白，Western blotting检测蛋白互作情况。
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