Cell-type specificity in the biotrophic interaction of Ustilago maydis and its host plant maize

生物营养真菌玉米黑粉菌侵染其寄主玉米的所有地上部分，在那里它会导致大的植物肿瘤的形成。这个项目建立在我们最初的假设之上，即梅迪斯杆菌部署器官特定的一组分泌的效应蛋白，以在不同的宿主组织中引起肿瘤。在前一个项目阶段，我们验证了这一想法，并确定了一组器官特异性效应器，这是玉米叶片中形成肿瘤所必需的，但在花序中不是。其中一个效应物See1被发现是诱导玉米叶片DNA合成所必需的，这种合成在肿瘤形成开始时被真菌重新激活。对感染玉米的叶片进行了详细的细胞学分析，发现玉米单胞菌诱导的叶片肿瘤是由两种不同的细胞过程形成的。束鞘细胞是增生性肿瘤细胞(HPTs)的起源，而叶肉细胞则转化为增生性肿瘤细胞(HTTS)。值得注意的是，See1效应器是细胞周期重新激活的重要诱导者，特别是在束鞘细胞中。用激光捕获显微切割技术从HTTS和HPTs以及See1缺失突变体诱导的HPTs中提取RNA进行转录组分析。该方法揭示了棉铃虫效应器基因的细胞型特异性基因表达。此外，它还提供了一组9个肥大相关效应因子的候选者。通过对玉米黑粉菌和不能诱发叶片肿瘤的密切相关病原菌雷氏孢子菌的比较转录组分析，发现差异转录调控与叶片和细胞型特异性效应因子的功能特别相关。基于这些结果，本项目旨在了解玉米黑粉菌诱导肿瘤形成的细胞型特异性机制。在确定See1是诱导植物细胞增殖所必需的效应器之后，我们现在的目标是剖析梅迪斯杆菌如何诱导肥大。为此，我们将鉴定并鉴定玉米单胞菌的肥大诱导效应因子(S)。我们将确定这些效应器(S)的植物靶标，以了解玉米粉虱诱导肥大的分子基础。最后，我们提出了一种新的方法，通过CRISPR-Cas9介导的基因替换方法，在瑞氏葡萄球菌中对肥大/肿瘤诱导效应进行功能分析，以期为病原体触发的真菌效应诱导肥大提供新的见解。最终，我们的策略旨在定义一个能够在植物中重建肿瘤形成的功能效应集，这将有助于我们在机制水平上理解这一令人兴奋的过程。

The biotrophic fungus Ustilago maydis infects all aerial parts of its host plant maize, where it causes the formation of large plant tumors. This project builds on our initial hypothesis that U. maydis deploys organ-specific sets of secreted effector proteins to cause tumors in the different host tissues. During the previous project phase we verified this idea and identified a set of organ-specific effectors, which are required for formation of tumors in maize leaves, but not in the inflorescences. One of these effectors, See1, was found to be required for the induction of DNA synthesis in maize leaves, which is re-activated by the fungus at the onset of tumor formation. Detailed cytological analyses of infected maize leaves revealed that U. maydis induced leaf tumors are formed by two distinct cellular processes. The bundle sheath cells are the origin of hyperplasic tumor cells (HPTs), while the mesophyll is transformed into hypertrophic tumor cells (HTTs). Strikingly, the See1 effector acts as an essential inducer of the cell cycle reactivation specifically in the bundle sheath cells. Laser-capture microdissection was used to isolate RNA for transcriptome analysis from HTTs and HPTs, as well as HPTs induced by the see1 deletion mutant. This approach revealed cell-type specific gene expression of U. maydis effector gene. Moreover, it provided a set of nine candidates for hypertrophy-related effectors. A comparative transcriptome analysis of U. maydis with the closely related pathogen Sporisorium reilianum, which cannot induce leaf tumors, revealed differential transcriptional regulation being particularly relevant for the function of leaf- and cell-type specific effectors.Based on these results, this project aims to understand the cell-type specific mechanisms underlying U. maydis induced tumor formation. After having identified See1 as an effector being required for the induction of plant cell proliferation, we now aim to dissect how U. maydis induces hypertrophy. To this end, we will identify and functionally characterize the hypertrophy-inducing effector(s) of U. maydis. We will identify the plant targets of these effector(s) to understand the molecular basis of hypertrophy induction by U. maydis. Finally, we proposed a novel approach to functionally analyze hypertrophy-/tumor-inducing effectors by a CRISPR-Cas9 mediated gene replacement approach in S. reilianum.We expect this project to provide novel insights into the pathogen-triggered induction of hypertrophy by fungal effectors. Ultimately, our strategy aims to define a functional effector-set able to reconstruct tumor formation in plants, which will help us to understand this exciting process on the mechanistic level.