稻瘟菌NLP蛋白家族的生物学功能及作用机理研究

Nep1-(necrosis- and ethylene-inducing protein 1) like proteins (NLPs) constitute a family of proteins widely distributed in many plant-associated microbes including bacteria, fungi and oomycetes. NLPs are noncytotoxic to monocotyledonous plants, although many of them cause necrosis on dicotyledons. It has been shown that NLPs are closely involved in establishment of infection and development of disease; however, little is known about the roles that these proteins and especially the noncytotoxic NLPs play during pathogenesis. Our initial investigation on rice blast fungus revealed that (1) all 4 members of the Nlp gene family are highly conserved among various field isolates; (2) transcript levels of these genes were significantly upregulated during infection of the rice plant; (3) individual members of the family may complement the loss-of-function of other members; and (4) knocking out each Nlp gene progressively led to the reduction of virulence of the pathogen. We therefore deduce that the NLP protein family is involved in pathogenesis of the blast disease and has an essential biological role in Magnaporthe oryzae. Based on these findings, we propose a broader investigation on this family of proteins in various fugal biological processes including growth and development, pathogenesis, and stress responses. By using transcriptomic and metabolomic assays combined with the efforts on searching for NLP-interacting molecules, we aim to reveal the mechanisms underlying the biological role of NLP protein family in the pathogen and to provide valuable information for development of novel strategy of disease control.

Nep1类似蛋白（NLPs）是广泛存在于多种植物病原物中的一个蛋白家族。虽然许多NLP蛋白对双子叶植物细胞具有致死毒性，但对单子叶植物没有毒性。已有研究表明NLP蛋白与病菌侵染过程密切相关，但人们对这类蛋白，尤其是非毒性NLP在致病过程中的作用了解很少。前期研究中我们发现：（1）稻瘟菌Nlp基因家族在不同菌株间高度保守；（2）这些Nlp基因在稻瘟菌侵染水稻时显著上调；（3）某些Nlp成员可以补偿其它成员的缺失；（4）逐步敲除Nlp基因可导致稻瘟菌致病力减弱。因此，我们推断NLP蛋白家族参与稻瘟菌致病过程，对稻瘟菌具有重要的生物学意义。本项目将以此为基础，全面研究NLP蛋白家族在稻瘟菌生长发育、侵染循环以及逆境响应等过程中的作用，并通过转录组和代谢组分析，结合与NLP蛋白互作分子的筛选与鉴定，深入解析该蛋白家族的生物学功能与作用的机理，为研究稻瘟病控制策略提供有益的参考。

NLP蛋白是一类微生物相关分子模式，广泛分布于不同的微生物类群。目前除了已知其对双子叶植物的细胞有毒性以外，我们对此类蛋白在微生物的生长和发育、响应环境胁迫以及与植物的相互作用等生物学过程中发挥的作用知之甚少。本研究首先分析了不同条件下稻瘟菌MoNLP基因的转录表达特征。结果表明，在侵染感病水稻的过程中，MoNLP基因被诱导表达，但各个基因的上调表达进程明显不同。我们还观察到，当用不同培养基或高盐及高渗培养基培养稻瘟菌时，MoNLP基因上调表达模式也存在显著差异。这些观察表明MoNLP的功能可能因响应环境因子不同而在功能上存在分化。为了进一步研究MoNLP家族的生物学功能，我们构建了无标记基因敲除载体，并用它敲除了稻瘟菌MoNLP家族的所有四个基因。通过比较野生型和MoNLP缺陷型菌株在侵染水稻过程中的毒性和生长情况，未观察到二者的致病力水平存在显著差异。同样，两种菌株在平板上的产孢和菌落生长水平相当，在营养不良和非生物胁迫条件下，如高盐、渗透和干旱等各种不利培养条件下生长水平也相似。这些数据表明，稻瘟菌并非必需MoNLP家族参与抵御这些不利的环境条件，尤其对于稻瘟菌侵染水稻也是非必需的。最后，我们观察到MoNLP1对拟南芥的细胞毒性存在明显的自然变异，通过QTL分析，我们确定了在植物中介导MoNLP1诱导细胞死亡的遗传位点。通过对该位点进行分子克隆，鉴定得到一个编码LRR蛋白的基因，该蛋白可与MoNLP1发生直接相互作用，并促进其寡聚化，从而促进MoNLP1诱导细胞死亡。本研究为NLP的细胞毒性模式提供了新的依据，并为进一步解析由MoNLP介导的生物学过程的的机制奠定了基础。