利用转座子Tn5诱变植物青枯菌获得胞外蛋白输出功能丧失突变体

 通过Tn5诱变技术,分离了植物青枯菌生理小种1号、3号的胞外蛋白输出功能丧失突变体。由于Tn5在其基因组单一的eep位点的插入,突变体失去了向培养滤液分泌产生胞外酶和胞外蛋白质的能力。用碱性磷酸酯酶基因PhoA作为报导基因,研究这些胞外蛋白穿越突变体细胞内膜,结果表明,这些胞外蛋白质可以穿过其内膜,但失去了穿越其外膜的能力。胞外多糖在植物体内和体外的产生没有受到eep基因位点突变的影响。该突变体失去了对番茄植株的致萎能力。

ON THE EXTROCELLULLAR PROTEINS DEFECTIVE MUTANT OF PSEUDOMONAS SOLANACEARUM GENERATED BY Tn5 MUTAGENESIS

We have isolated mutants of race 1 and race 3 strains of P. solanacearum with Tn5 insertions at a single locus(eep) whose culture supernatants lack all of its known extracellular enzymes and most other detectable extracellular proteins(EXPs). Analysis of subcellu-lar fractions of eep::Tn5 mutants showed that they still synthesize many of these EXPs, but accumulated them inside the cell implying that eep is involved in protein export. These analyses and others with PhoA fusion proteins showed that export of proteins across the inner membrane is not affected by the eep mutation, suggesting that the eep locus functions only in protein export across the outer membrane. However, neither production nor export of extracellular polysaccharide was obviously affected by the eep mutation. Analysis of the in planta behavior of eep mutants after stem-inoculation into tomato plants showed that they had lost the ability to cause wilt symptoms or kill the plant, possibly because they colonized plant stems much more slowly than wild type strains. Plants grown in soil inoculated with the mutants did not develop any visible disease symptoms over a 20-d period and their stems showed no evidence of infection by P. solanacearum cells. Under the same conditions wild types killed in 14 d and >109 cells were found in their stems. These results suggest that an individual or group of extracellular proteins of P. solanacearum are required for infection via the roots, as well as wilting and killing the host plants.