Bio-protection and Ecology Division, P.O. Box 84, Lincoln University, Canterbury, New Zealand.
Mycologia. 2004 Nov-Dec;96(6):1245-52.
Mycoparasitism of fungal plant pathogens by Trichoderma species is a complex process that involves the production and coordinated secretion of cell-wall degrading enzymes. Genes implicated in mycoparasitism by Trichoderma atroviride contain motifs in the promoter region, designated MYRE1-MYRE4, that are proposed to act as binding sites for a global inducer of the mycoparasitic response. The aim of our study was to establish whether these motifs also were present in Trichoderma hamatum and whether the presence of these motifs could predict co-expression when T. hamatum was confronted by a pathogen. Using a combination of targeted, degenerate and inverse PCR, homologues of the mycoparasitism-related genes ech42 (chit42), prb1 and lam1.3 (xbg1.3-110), which encode an endochitinase, proteinase, and β-1,3-glucanase, respectively, were cloned and sequenced from T. hamatum. Alignment of the promoter regions of the three genes revealed identical regions in the chit42 and prb1 promoters, which were 6-9 base pairs in length and conserved in position. Specifically, the regulator y motifs MYRE1-MYRE4 were fully conserved, together with a fifth motif, identified by this research. A substrate assay designed to investigate the response of these genes from T. harzianum and T. hamatum to a simple carbon source (glycerol) showed that, in contrast to chit42 and prb1, xbg1.3-110 was not expressed. Further comparison of the expression patterns of these three genes between T. harzianum and T. hamatum using the glycerol substrate assay showed that no chit42 or prb1 expression could be detected in T. harzianum when it was grown under the same conditions as T. hamatum. This showed that the response of these genes to glycerol was species specific and that a single expression pattern for these genes was not common to all Trichoderma species. Confrontation assays were used to investigate the response of the three T. hamatum genes to the more complex substrate posed by the fungal pathogen Sclerotinia sclerotiorum. Once again gene expression analysis showed that both chit42 and prb1 were co-expressed and moderately induced during confrontation against Sclerotinia sclerotiorum. Although xbg1.3-110 previously had been implicated in mycoparasitism by T. harzianum, this study detected no xbg1.3-110 expression during confrontation between T. hamatum and S. sclerotiorum. These findings show that the MYRE1-MYRE4 together with MYRE5 are present in two species of Trichoderma, T. atroviride and T. hamatum and that the presence of these motifs could predict co-expression in response to two carbon sources.
木霉属真菌对植物病原菌的寄生作用是一个复杂的过程，涉及到细胞壁降解酶的产生和协调分泌。涉及木霉属真菌寄生作用的基因在启动子区域包含有 MYRE1-MYRE4 特征基序，这些基序被提议作为寄生反应的全局诱导物的结合位点。我们的研究目的是确定这些基序是否也存在于哈茨木霉中，以及当哈茨木霉遇到病原体时，这些基序的存在是否可以预测共表达。使用靶向、简并和反向 PCR 的组合，从哈茨木霉中克隆并测序了与寄生作用相关的基因 ech42（几丁质 42）、prb1 和 lam1.3（xbg1.3-110）的同源物，它们分别编码内切几丁质酶、蛋白酶和β-1,3-葡聚糖酶。对三个基因启动子区域的序列比对显示，chit42 和 prb1 启动子中有相同的区域，长度为 6-9 个碱基对，位置保守。具体来说，调节剂 y 基序 MYRE1-MYRE4 完全保守，同时还发现了一个由本研究确定的第五个基序。设计了一个底物测定法来研究这些基因在哈茨木霉和木霉中的反应，结果表明，与 chit42 和 prb1 不同，xbg1.3-110 没有表达。使用甘油底物测定法进一步比较哈茨木霉和哈茨木霉这三个基因的表达模式表明，当哈茨木霉在与哈茨木霉相同的条件下生长时，无法检测到 chit42 或 prb1 的表达。这表明这些基因对甘油的反应是种特异性的，并且这些基因的单一表达模式并不适用于所有木霉属物种。对峙试验用于研究哈茨木霉的三个基因对真菌病原体核盘菌更复杂的底物的反应。基因表达分析再次表明，chit42 和 prb1 在与核盘菌对峙时均共表达并适度诱导。尽管 xbg1.3-110 先前被认为与哈茨木霉的寄生作用有关，但本研究在哈茨木霉与核盘菌的对峙中未检测到 xbg1.3-110 的表达。这些发现表明，在两个木霉属物种，即深绿木霉和哈茨木霉中，存在 MYRE1-MYRE4 以及 MYRE5，并且这些基序的存在可以预测对两种碳源的共表达反应。
