Alfano J R, Klm H S, Delaney T P, Collmer A
Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA.
Mol Plant Microbe Interact. 1997 Jul;10(5):580-8. doi: 10.1094/MPMI.1997.10.5.580.
A 25-kb DNA region, previously cloned from Pseudomonas syringae pv. syringae 61 in cosmid pHIR11, enables nonpathogenic bacteria such as Pseudomonas fluorescens and Escherichia coli to elicit the hypersensitive response (HR) in tobacco (Nicotiana tabacum). hrmA is located within this region, adjacent to a conserved cluster of hrp genes, and is essential for nonpathogens to elicit the HR. DNA sequence analysis suggested that hrmA was the second of two genes in an operon and was preceded by an open reading frame (ORF), ORF1, which is predicted to encode a 10.9-kDa protein. DNA gel blot analysis revealed that sequences hybridizing with a DNA fragment internal to hrmA were absent from P. syringae pv. syringae B728a, P. syringae pv. tabaci 11528, and P. syringae pv. glycinea race 4 U1, but present in P. syringae pv. tomato DC3000. A 2.4-kb BamHI-AvrII fragment carrying hrmA, ORF1, and native regulatory sequences was subcloned into broad-host-range vector pDSK519 and electroporated into P. syringae pv. syringae B728a and P. syringae pv. tabaci 11528. The presence of the hrmA locus had no apparent effect on the ability of P. syringae pv. syringae B728a to cause brown spot of bean, but it caused P. syringae pv. tabaci 11528 to elicit the defense-associated HR rather than disease in N. tabacum cvs. Xanthi N and Xanthi NC and N. clevelandii. Furthermore, N. debeyii, N. glutinosa, N. rustica, and N. tabacum cvs. Petit Havana and Samsun responded with the HR to P. fluorescens(pHIR11). In contrast, N. benthamiana-P. syringae pv. tabaci interactions were unaffected by the presence of HrmA, and P. fluorescens(pHIR11) did not elicit the HR in N. benthamiana. The hrmA ORF was subcloned into pFLAG-CTC, which expressed HrmA with a C-terminal FLAG synthetic epitope fusion. Escherichia coli MC4100 cells carrying the functional hrp cluster and the hrmA-FLAG derivative secreted the HrpZ harpin, but not HrmA-FLAG, to the medium, as indicated by immunoblot analysis with M2 anti-FLAG and polyclonal anti-HrpZ antibodies. The hrmA ORF was also subcloned into plant expression vector pFF19 and then biolistically delivered, along with pFF19G (expressing beta-glucuronidase), into suspension-cultured tobacco cells. Histochemical staining 24 h later revealed substantial beta-glucuronidase activity in cells receiving pFF19G and pFF19 but not in those receiving pFF19G and pFF19-HrmA. Thus, internal production of HrmA was deleterious to tobacco cells.
一个25千碱基对的DNA区域，先前从丁香假单胞菌丁香致病变种61中克隆到黏粒pHIR11中，它能使诸如荧光假单胞菌和大肠杆菌等非致病细菌在烟草（烟草属）中引发超敏反应（HR）。hrmA位于该区域内，与hrp基因的保守簇相邻，对于非病原体引发超敏反应至关重要。DNA序列分析表明，hrmA是操纵子中两个基因中的第二个，其前面有一个开放阅读框（ORF），即ORF1，预计它编码一个10.9千道尔顿的蛋白质。DNA凝胶印迹分析显示，与hrmA内部的一个DNA片段杂交的序列在丁香假单胞菌丁香致病变种B728a、烟草假单胞菌11528和大豆假单胞菌小种4 U1中不存在，但在番茄假单胞菌DC3000中存在。一个携带hrmA、ORF1和天然调控序列的2.4千碱基对的BamHI - AvrII片段被亚克隆到广宿主范围载体pDSK519中，并通过电穿孔导入丁香假单胞菌丁香致病变种B728a和烟草假单胞菌11528中。hrmA基因座的存在对丁香假单胞菌丁香致病变种B728a引起豆类褐斑的能力没有明显影响，但它使烟草假单胞菌11528在烟草品种Xanthi N、Xanthi NC和克利夫兰烟草中引发与防御相关的超敏反应而不是疾病。此外，德贝烟草、黏性烟草、黄花烟草以及烟草品种Petit Havana和Samsun对荧光假单胞菌（pHIR11）产生超敏反应。相比之下，本氏烟草 - 烟草假单胞菌的相互作用不受HrmA存在的影响，并且荧光假单胞菌（pHIR11）在本氏烟草中不引发超敏反应。hrmA开放阅读框被亚克隆到pFLAG - CTC中，该载体表达带有C末端FLAG合成表位融合的HrmA。携带功能性hrp簇和hrmA - FLAG衍生物的大肠杆菌MC4100细胞将HrpZ激发子分泌到培养基中，但不分泌HrmA - FLAG，用M2抗FLAG和多克隆抗HrpZ抗体进行免疫印迹分析表明了这一点。hrmA开放阅读框也被亚克隆到植物表达载体pFF19中，然后与pFF19G（表达β - 葡萄糖醛酸酶）一起通过生物弹道法导入悬浮培养的烟草细胞中。24小时后的组织化学染色显示，在接受pFF19G和pFF19的细胞中有大量的β - 葡萄糖醛酸酶活性，而在接受pFF19G和pFF19 - HrmA的细胞中则没有。因此，HrmA在细胞内的产生对烟草细胞有害。
