Valette-Collet Odile, Cimerman Agnès, Reignault Philippe, Levis Caroline, Boccara Martine
Pathologie Végétale UMR217, Institut National Agronomique 75231 Paris, France.
Mol Plant Microbe Interact. 2003 Apr;16(4):360-7. doi: 10.1094/MPMI.2003.16.4.360.
The pectinolytic enzyme pectin methylesterase (PME) hydrolyses pectin in methanol and polygalacturonic acid. In the expressed sequence tag library of Botrytis cinerea T4, we identified a 1,041 bp Bcpme1 cDNA potentially encoding a 346-amino acid protein of 37 kDa showing 46.8% identity with Aspergillus sp. PMEs. Bcpme1 is a single copy gene and is similarly expressed in glucose and pectin containing media. To evaluate the role of Bcpme1 in Botrytis cinerea virulence, a mutant in Bcpme1 was generated by gene disruption. The Bcpme1 mutant showed similar growth on rich medium but reduced growth on pectin medium. Two isozymes of pI 7.4 and 7.1 were detected in pectin liquid-culture supernatants of wild-type strain Bd90 analyzed by isoelectric focusing-polyacrylamide gel electrophoresis, while those of Bcpme1 mutant possessed only the pI 7.1 isozyme. BCPME1, the pI 7.4 isozyme, is the major PME activity, as PME activity is 75% reduced in Bcpme1 mutant. Moreover, the Bcpme1 mutant was less virulent on apple fruits, grapevine, and Arabidopsis thaliana leaves. Those phenotypes were complemented by reintroducing a Bcpme1 copy in the Bcpme1 mutant. These results showed that B. cinerea possessed more than one PME-encoding gene and that BCPME1 is an important determinant of B. cinerea virulence.
果胶分解酶果胶甲酯酶（PME）可将果胶水解为甲醇和聚半乳糖醛酸。在灰葡萄孢菌T4的表达序列标签文库中，我们鉴定出一个1041 bp的Bcpme1 cDNA，它可能编码一个37 kDa的346个氨基酸的蛋白质，与曲霉属的PMEs有46.8%的同一性。Bcpme1是一个单拷贝基因，在含有葡萄糖和果胶的培养基中表达相似。为了评估Bcpme1在灰葡萄孢菌毒力中的作用，通过基因敲除产生了Bcpme1突变体。Bcpme1突变体在丰富培养基上生长相似，但在果胶培养基上生长减少。通过等电聚焦-聚丙烯酰胺凝胶电泳分析野生型菌株Bd90的果胶液体培养上清液，检测到两种pI分别为7.4和7.1的同工酶，而Bcpme1突变体的同工酶仅具有pI 7.1的同工酶。pI 7.4的同工酶BCPME1是主要的PME活性，因为Bcpme1突变体中的PME活性降低了75%。此外，Bcpme1突变体在苹果果实、葡萄藤和拟南芥叶片上的毒力较低。通过在Bcpme1突变体中重新引入一个Bcpme1拷贝，这些表型得到了互补。这些结果表明，灰葡萄孢菌拥有多个编码PME的基因，并且BCPME1是灰葡萄孢菌毒力的一个重要决定因素。
