Plant pathogens utilize a portfolio of secreted effectors to successfully infect and manipulate their hosts. It is, however, still unclear whether changes in secretomes leading to host specialization involve mostly effector gene gains/losses or changes in their sequences. To test these hypotheses, we compared the secretomes of three host-specific castrating anther smut fungi (Microbotryum), two being sister species. To address within-species evolution, which might involve coevolution and local adaptation, we compared the secretomes of strains from differentiated populations. We experimentally validated a subset of signal peptides. Secretomes ranged from 321 to 445 predicted secreted proteins (SPs), including a few species-specific proteins (42 to 75), and limited copy number variation, i.e., little gene family expansion or reduction. Between 52% and 68% of the SPs did not match any Pfam domain, a percentage that reached 80% for the small secreted proteins, indicating rapid evolution. In comparison to background genes, we indeed found SPs to be more differentiated among species and strains, more often under positive selection, and highly expressed in planta; repeat-induced point mutations (RIPs) had no role in effector diversification, as SPs were not closer to transposable elements than background genes and were not more RIP affected. Our study thus identified both conserved core proteins, likely required for the pathogenic life cycle of all Microbotryum species, and proteins that were species specific or evolving under positive selection; these proteins may be involved in host specialization and/or coevolution. Most changes among closely related host-specific pathogens, however, involved rapid changes in sequences rather than gene gains/losses.IMPORTANCE Plant pathogens use molecular weapons to successfully infect their hosts, secreting a large portfolio of various proteins and enzymes. Different plant species are often parasitized by host-specific pathogens; however, it is still unclear whether the molecular basis of such host specialization involves species-specific weapons or different variants of the same weapons. We therefore compared the genes encoding secreted proteins in three plant-castrating pathogens parasitizing different host plants, producing their spores in plant anthers by replacing pollen. We validated our predictions for secretion signals for some genes and checked that our predicted secreted proteins were often highly expressed during plant infection. While we found few species-specific secreted proteins, numerous genes encoding secreted proteins showed signs of rapid evolution and of natural selection. Our study thus found that most changes among closely related host-specific pathogens involved rapid adaptive changes in shared molecular weapons rather than innovations for new weapons.

中文翻译：

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原因和影响因素：全基因组比较揭示了宿主特异性植物破坏性真菌之间共享但迅速发展的影响因素集合。

植物病原体利用分泌的效应子组合成功感染并操纵其宿主。但是，尚不清楚导致宿主特化的分泌组的变化是否主要涉及效应基因的增/减或其序列的变化。为了检验这些假设，我们比较了三种宿主特异性cast割花药黑穗病真菌（Microbotryum）的分泌组，其中两种是姊妹物种。为了解决可能涉及协同进化和局部适应的物种内进化，我们比较了来自不同种群的菌株的分泌组。我们通过实验验证了信号肽的子集。分泌组的预测分泌蛋白（SP）范围为321至445，其中包括一些物种特异性蛋白（42至75），并且拷贝数变化有限，即几乎没有基因家族扩展或减少。52％到68％的SP与任何Pfam结构域都不匹配，小分泌蛋白的百分比达到80％，表明进化迅速。与背景基因相比，我们确实发现SP在物种和品系之间具有更高的区分度，通常在正选择下，并且在植物中高表达。重复诱导的点突变（RIP）在效应子的多样化中没有作用，因为SP与背景基因相比，离转座因子更近，并且不受RIP影响更大。因此，我们的研究既确定了保守的核心蛋白，可能是所有微型菌的致病生命周期所必需的，又是特定于物种或在正选择下进化的蛋白。这些蛋白质可能参与宿主的专门化和/或协同进化。在密切相关的宿主特定病原体中，大多数变化 重要的是，植物病原体利用分子武器成功感染宿主，并分泌大量蛋白质和酶。宿主特有的病原体常常使不同的植物种类寄生。但是，目前尚不清楚这种宿主专门化的分子基础是否涉及特定物种的武器或同一武器的不同变体。因此，我们比较了寄生在不同寄主植物中的三种植物播种病原体中编码分泌蛋白的基因，通过取代花粉在植物花药中产生了孢子。我们验证了我们对某些基因分泌信号的预测，并检查了我们预测的分泌蛋白在植物感染期间通常高表达。尽管我们发现的物种特异性分泌蛋白很少，但编码该分泌蛋白的众多基因却显示出快速进化和自然选择的迹象。因此，我们的研究发现，在密切相关的特定宿主病原体中，大多数变化涉及共享分子武器的快速适应性变化，而不是新武器的创新。