Jenner C, Hitchin E, Mansfield J, Walters K, Betteridge P, Teverson D, Taylor J
Department of Biochemistry and Biological Sciences, Wye College, Ashford, Kent, U.K.
Mol Plant Microbe Interact. 1991 Nov-Dec;4(6):553-62.
The gene for cultivar-specific avirulence to Phaseolus vulgaris cv. Tendergreen in races 3 and 4 of Pseudomonas syringae pv. phaseolicola was isolated and sequenced. Genomic clones from libraries of race 3 in pLAFR1 and race 4 in pLAFR3, which altered the phenotype of race 5 from virulent to avirulent in Tendergreen, were found to possess a common approximately 15-kb region of DNA that contained the determinant of avirulence. Subcloning and insertion mutagenesis with Tn1000 located an avirulence gene within a 1.4-kb BglII/HindIII DNA fragment in races 3 and 4. Comparison of the nucleotide sequences of regions of DNA that confer avirulence confirmed that both races have an identical gene for avirulence (designated avrPph3) comprising 801 base pairs (bp) and predicted to encode a cytoplasmic protein of 28,703 Da. A sequence, TGCAACCGAAT, 91% homologous to the motif found in promoter regions of avrB and avrD from P. s. pv. glycinea was located 89-99 bp upstream of the start of the open-reading frame 1. Hybridization experiments showed that avrPph3 was not plasmid-borne and was absent from isolates of P. s. pv. phaseolicola races 1, 2, 5, 6, 7, and 8, P. cichorii, P. s. pvs. coronafaciens, glycinea, maculicola, pisi, syringae, and tabaci. Cosegregation studies of crosses between cultivars resistant (Tendergreen) and susceptible (Canadian Wonder) to races 3 and 4 and transconjugants of race 5 confirmed that a gene-for-gene relationship controls specificity in the interaction between Tendergreen and races 3 and 4 of P. s. pv. phaseolicola.
分离并测定了丁香假单胞菌菜豆致病变种3和4中对菜豆品种嫩绿色具有品种特异性无毒力的基因。从pLAFR1载体的3号生理小种文库和pLAFR3载体的4号生理小种文库中筛选出的基因组克隆，能使5号生理小种在嫩绿色品种上的表型从有毒变为无毒，这些克隆被发现含有一个约15kb的共同DNA区域，该区域包含无毒力决定因子。利用Tn1000进行亚克隆和插入诱变，在3号和4号生理小种的一个1.4kb BglII/HindIII DNA片段中定位到一个无毒力基因。对赋予无毒力的DNA区域的核苷酸序列进行比较，证实这两个生理小种具有相同的无毒力基因（命名为avrPph3），该基因由801个碱基对（bp）组成，预计编码一个28703Da的细胞质蛋白。在开放阅读框1起始位点上游89 - 99bp处，发现了一个与大豆丁香假单胞菌avrB和avrD启动子区域中基序有91%同源性的序列TGCAACCGAAT。杂交实验表明，avrPph3不是质粒携带的，并且在菜豆丁香假单胞菌1、2、5、6、7和8号生理小种、菊苣假单胞菌、丁香假单胞菌的其他致病变种（如冠腐致病变种、大豆致病变种、黄斑致病变种、豌豆致病变种、丁香致病变种和烟草致病变种）的分离物中不存在。对嫩绿色（抗病）和加拿大奇迹（感病）品种与3号和4号生理小种的杂交后代以及5号生理小种的转接合子进行共分离研究，证实了基因对基因关系控制着嫩绿色品种与菜豆丁香假单胞菌3号和4号生理小种相互作用中的特异性。
