Biocentre Helsinki, Department of Biosciences, Division of General Microbiology, PO Box 56, FIN-00014 University of Helsinki, Finland.
Mol Plant Pathol. 2002 Nov 1;3(6):451-60. doi: 10.1046/j.1364-3703.2002.00139.x.
SUMMARY Pseudomonas syringae pv. tomato is the causative agent of bacterial speck of tomato. The key virulence determinant of P. syringae is the hrp gene cluster, which encodes a type III secretion system. The type III system is used by a wide variety of pathogenic bacteria for transporting virulence proteins from the bacteria directly into the eukaryotic host cell. Hrp pilus, which is composed of HrpA pilin subunits, is an indispensable component of the type III secretion system in P. syringae. Here we have determined the spatial and temporal expression pattern of hrpA of P. syringae DC3000 in intact leaves, using a HrpA-GFP protein fusion and confocal microscopy. The hrpA gene was strongly and rapidly induced inside the leaf tissues after infiltration of the bacteria. After spray-inoculation, hrpA-induced bacteria were detected endophytically 72 h post-inoculation, and 96 h after spray-inoculation, disease symptoms appeared and GFP-expressing bacteria were observed at symptom sites, both endo- and epiphytically. Live/dead staining of the bacteria showed that Pst DC3000 does not survive well on leaf surfaces. Apoplastic populations were apparently bursting on to the leaf surface through stomata. Kinetics of population sizes of wild-type DC3000 and hrpA(-) showed significant differences, initially endophytically and only later epiphytically. Our results suggest that the Hrp pilus is first induced in the apoplast and apparently functions mainly inside the leaf tissues. These results suggest that P. syringae DC3000 mainly multiplies endophytically.
丁香假单胞菌 pv. 番茄是番茄细菌性斑点病的病原体。丁香假单胞菌的关键毒力决定因素是 hrp 基因簇，它编码一种 III 型分泌系统。III 型系统被多种病原菌用于将毒力蛋白从细菌直接输送到真核宿主细胞中。Hrp 菌毛由 HrpA 菌毛亚基组成，是丁香假单胞菌 III 型分泌系统的不可或缺组成部分。在这里，我们使用 HrpA-GFP 蛋白融合和共焦显微镜，确定了完整叶片中 DC3000 丁香假单胞菌的 hrpA 的时空表达模式。在细菌浸润后，hrpA 基因在叶片组织内迅速强烈诱导。喷雾接种后，72 小时后可检测到内生生菌 hrpA 诱导，96 小时后出现症状，在症状部位观察到 GFP 表达细菌，无论是内生的还是附生的。细菌的死活染色表明 Pst DC3000 在叶片表面的存活率不高。通过气孔，细胞外种群显然爆裂到叶片表面。野生型 DC3000 和 hrpA(-)的种群大小动力学表现出显著差异，最初是内生的，后来才是附生的。我们的结果表明，Hrp 菌毛首先在细胞外区诱导，显然主要在叶片组织内发挥作用。这些结果表明，丁香假单胞菌 DC3000 主要内生繁殖。
