Colletotrichum orbiculare FAM1 Encodes a Novel Woronin Body-Associated Pex22 Peroxin Required for Appressorium-Mediated Plant Infection.
Colletotrichum orbiculare FAM1 Encodes a Novel Woronin Body-Associated Pex22 Peroxin Required for Appressorium-Mediated Plant Infection.
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DOI：
10.1128/mbio.01305-15
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发表时间：
2015-09-15
期刊：
影响因子：
6.4
通讯作者：
O'Connell R
中科院分区：
文献类型：
--
作者：
Kubo Y;Fujihara N;Harata K;Neumann U;Robin GP;O'Connell R
黄瓜炭疽病真菌Colletotrichum orbiculare形成称为附着胞的特化细胞用于宿主渗透。我们确定了一个基因，FAM 1，编码一种新的过氧化物酶蛋白，是必不可少的过氧化物酶体生物合成和相关的Woronin机构（WBs），致密的核心囊泡，发现只有在丝状子囊真菌的功能，以保持细胞的完整性。fam 1破坏的突变体不能生长在含有油酸作为唯一碳源的培养基上，并且是非致病性的，在附着胞黑化和宿主穿透方面都有缺陷。携带过氧化物酶体靶向信号（PTS）的荧光蛋白没有输入到fam 1突变体的过氧化物酶体中，表明FAM 1是一种新的过氧化物酶体生物合成基因（peroxin）。FAM 1没有表现出显着的同源性，任何酿酒酵母过氧化物酶，但类似于保守的丝状子囊菌特异性Pex 22样蛋白，其中包含一个预测的Pex 4结合位点，并可能参与回收PTS受体从过氧化物酶体的胞质溶胶。圆形梭菌FAM 1补充了酿酒酵母pex 22突变体的过氧化物酶体基质蛋白输入缺陷。Fam 1-GFP（绿色荧光蛋白）融合蛋白的共聚焦显微镜和抗Fam 1抗体的免疫电镜显示Fam 1定位于从过氧化物酶体出芽的新生WB和成熟WB。通过与WB基质蛋白CoHex 1（圆形念珠菌Hex 1）和WB膜蛋白CoWsc（圆形念珠菌Wsc）共定位，并通过亚细胞分级分离和使用Fam 1和CoHex 1抗体的蛋白质印迹法证实Fam 1与WB的关联。在WB缺陷的cohex 1突变体中，Fam 1被重定向到过氧化物酶体膜。我们的研究结果表明，Fam 1是一个WB相关的过氧化物酶所需的发病机制，并提高了局部受体回收发生在WB的可能性。西瓜炭疽病菌是葫芦科植物上的一种重要病害。在本文中，我们描述了一种新的过氧化物酶体生物合成基因，从这种病原体称为FAM 1。虽然在酿酒酵母中没有具有显著同源性的基因，但FAM 1含有预测的Pex 22蛋白典型的Pex 4结合位点，其在PTS受体从过氧化物酶体到胞质溶胶的再循环中起作用。我们表明，FAM 1补充缺陷的过氧化物酶体基质蛋白进口的酿酒酵母pex 22突变体和fam 1突变体是完全缺陷的过氧化物酶体功能，脂肪酸代谢和致病性。值得注意的是，我们发现，这种新的peroxin是专门定位于边界膜的Woronin机构，这是小过氧化物酶体衍生的细胞器独特的丝状子囊真菌，功能在隔膜孔堵塞。我们的研究结果表明，这些真菌已增选Woronin机构的本地化受体回收过程中的基质蛋白进口。
The cucumber anthracnose fungus Colletotrichum orbiculare forms specialized cells called appressoria for host penetration. We identified a gene, FAM1, encoding a novel peroxin protein that is essential for peroxisome biogenesis and that associates with Woronin bodies (WBs), dense-core vesicles found only in filamentous ascomycete fungi which function to maintain cellular integrity. The fam1 disrupted mutants were unable to grow on medium containing oleic acids as the sole carbon source and were nonpathogenic, being defective in both appressorium melanization and host penetration. Fluorescent proteins carrying peroxisomal targeting signals (PTSs) were not imported into the peroxisomes of fam1 mutants, suggesting that FAM1 is a novel peroxisomal biogenesis gene (peroxin). FAM1 did not show significant homology to any Saccharomyces cerevisiae peroxins but resembled conserved filamentous ascomycete-specific Pex22-like proteins which contain a predicted Pex4-binding site and are potentially involved in recycling PTS receptors from peroxisomes to the cytosol. C. orbiculare FAM1 complemented the peroxisomal matrix protein import defect of the S. cerevisiae pex22 mutant. Confocal microscopy of Fam1-GFP (green fluorescent protein) fusion proteins and immunoelectron microscopy with anti-Fam1 antibodies showed that Fam1 localized to nascent WBs budding from peroxisomes and mature WBs. Association of Fam1 with WBs was confirmed by colocalization with WB matrix protein CoHex1 (C. orbiculare Hex1) and WB membrane protein CoWsc (C. orbiculare Wsc) and by subcellular fractionation and Western blotting with antibodies to Fam1 and CoHex1. In WB-deficient cohex1 mutants, Fam1 was redirected to the peroxisome membrane. Our results show that Fam1 is a WB-associated peroxin required for pathogenesis and raise the possibility that localized receptor recycling occurs in WBs. Colletotrichum orbiculare is a fungus causing damaging disease on Cucurbitaceae plants. In this paper, we characterize a novel peroxisome biogenesis gene from this pathogen called FAM1. Although no genes with significant homology are present in Saccharomyces cerevisiae, FAM1 contains a predicted Pex4-binding site typical of Pex22 proteins, which function in the recycling of PTS receptors from peroxisomes to the cytosol. We show that FAM1 complements the defect in peroxisomal matrix protein import of S. cerevisiae pex22 mutants and that fam1 mutants are completely defective in peroxisome function, fatty acid metabolism, and pathogenicity. Remarkably, we found that this novel peroxin is specifically localized on the bounding membrane of Woronin bodies, which are small peroxisome-derived organelles unique to filamentous ascomycete fungi that function in septal pore plugging. Our finding suggests that these fungi have coopted the Woronin body for localized receptor recycling during matrix protein import.