Ralstonia solanacearum OE1-1 (OE1-1) induced necrotic lesions in an infiltrated area of tobacco leaves 72 h after infiltration, and the leaves had wilted at 5 days. Here we report phenotypes of the OE1-1 mutant deleted hrpB or hrpY with respect to colonization and proliferation in infiltrated tobacco leaves and the induction of host responses immediately after invasion. An hrpB-deleted mutant and an hrpY-deleted mutant grew similar to the parent strain, OE1-1, in vitro. When infiltrated into tobacco leaves, the mutants lost their ability to induce necrotic lesions and provoke the disease. Populations of the mutants in the infiltrated area were retained equally after infiltration, and the mutants were not detected in any other region. Transcripts of hsr203J and hin1, which are marker genes of plant-microbe interactions and were detected 8 h after infiltration of OE1-1 in the infiltrated area of tobacco leaves, were not detected in the mutant-infiltrated tobacco leaves. These results suggest that the hrp mutants, which are deficient in type III secretion machinery, lose their ability to colonize and multiply in host plants immediately after invasion, resulting in a loss of their ability to induce host responses and the subsequent provocation of disease.

Ralstonia solanacearum OE1-1 (OE1-1) is pathogenic to tobacco. The type III-secreted effector protein popA of OE1-1 showed 97.6% identity to popA of R. solanacearum GMI1000, which is not pathogenic to tobacco. Reverse transcription-polymerase chain reaction analysis showed that popA in OE1-1 was expressed at 3 h after inoculation (HAI), but not before, in infiltrated-tobacco leaves. Pathogenicity analysis using a popABC operon-deleted mutant of OE1-1 (deltaABC) showed that popABC is not directly involved in the pathogenicity of OE1-1. When Papa, which constitutively expresses popA, was infiltrated into tobacco leaves, popA was expressed by 0.5 HAI. Papa could no longer multiply or spread in tobacco leaves and was no longer virulent. Moreover, the hypersensitive response (HR) and expression of HR-related genes were not induced in Papa-infiltrated leaves. Papa was also avirulent in a tobacco root-dipping inoculation assay. These results suggest that the expression of popA in Papa immediately after invasion triggers the suppression of bacterial proliferation and movement, resulting in loss of virulence. However, Papa retained its virulence when directly inoculated into xylem vessels. This result suggests that tobacco plants can recognize PopA when it is expressed early in disease development, and respond with an effective defense in the intercellular spaces.

The Capsicum spp. L genes (L(1) to L(4)) confer resistance to tobamoviruses. Currently, the L(4) gene from Capsicum chacoense is the most effective resistance gene and has been used widely in breeding programs in Japan which have developed new resistant cultivars against Pepper mild mottle virus (PMMoV). However, in 2004, mild mosaic symptoms began appearing on the leaves of commercial pepper plants in the field which possessed the L(4) resistance gene. Serological and biological assays on Capsicum spp. identified the causal virus strain as a previously unreported pathotype, P(1,2,3,4). PMMoV sequence analysis of the virus and site-directed mutagenesis using a PMMoV-J of the P(1,2) pathotype revealed that two amino acid substitutions in the coat protein, Gln to Arg at position 46 and Gly to Lys at position 85, were responsible for overcoming the L(4) resistance gene.

All authors of the ten research groups contributed equally to this work. The authors of each research group are grouped together. Further information about the interactions between "Micro-Tom" and pathogens will be supplied at our web site (http://www.agri.tohoku.ac. jp/ppathol/tomato/). Abstract Lycopersicon esculentum cultivar Micro-Tom is a miniature tomato with many advantages for studies of the molecular biology and physiology of plants. To evaluate the suitability of Micro-Tom as a host plant for the study of pathogenesis, Micro-Tom plants were inoculated with 16 well-known fungal, bacterial, and viral pathogens of tomato. Athelia rolfsii, Botryotinia fuckeliana, Oidium sp., Phytophthora infestans, and Sclerotinia sclerotiorum caused typical symptoms and sporulated abundantly on MicroTom. Micro-Tom was resistant to Alternaria alternata, Corynespora cassiicola, and Fusarium oxysporum. When Micro-Tom was inoculated with 17 isolates of Ralstonia solanacearum, many isolates induced wilt symptoms. Agrobacterium tumefaciens also was pathogenic, causing crown galls on stem tissue after needle prick inoculation. In MicroTom sprayed with Pseudomonas syringae pv. tomato, P. s. pv. tabaci, or P. s. pv. glycinea, bacterial populations did not increase, and yellow lesions appeared only on leaves sprayed with P. s. pv. tomato. Tomato mosaic virus, Tomato aspermy virus, and Cucumber mosaic virus systemically infected Micro-Tom, which developed symptoms characteristic of other cultivars of tomato after infection with the respective virus. These results indicated that Micro-Tom was generally susceptible to most of the important tomato pathogens and developed typical symptoms, whereas certain pathogens were restricted by either hypersensitive resistance or nonhost resistance on Micro-Tom. Therefore, an assortment of Micro-Tom-pathogen systems should provide excellent models for studying the mechanism of susceptible and resistant interactions between plants and pathogens.

The virulence factor of Melon necrotic spot virus (MNSV), a virus that induces systemic necrotic spot disease on melon plants, was investigated. When the replication protein p29 was expressed in N. benthamiana using a Cucumber mosaic virus vector, necrotic spots appeared on the leaf tissue. Transmission electron microscopy revealed abnormal mitochondrial aggregation in these tissues. Fractionation of tissues expressing p29 and confocal imaging using GFP-tagged p29 revealed that p29 associated with the mitochondrial membrane as an integral membrane protein. Expression analysis of p29 deletion fragments and prediction of hydrophobic transmembrane domains (TMDs) in p29 showed that deletion of the second putative TMD from p29 led to deficiencies in both the mitochondrial localization and virulence of p29. Taken together, these results indicated that MNSV p29 interacts with the mitochondrial membrane and that p29 may be a virulence factor causing the observed necrosis.

A viroid disease causing chlorosis of leaves and dwarfism was found on commercial tomato plants in Hiroshima Prefecture, Japan. Grafting of stems from infected plants onto healthy plants resulted in the same symptoms on the healthy plants. Small RNAs were isolated from infected plant tissue and caused identical symptoms by 3-4 week after mechanical inoculation of tomato seedlings. Nucleotide sequencing indicated that the causal pathogen was Tomato chlorotic dwarf viroid (TCDVd) sharing 98% nucleotide sequence identity with that of a Canadian isolate reported previously. This description is the first of TCDVd infection of tomato plants in Japan.

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The vigorous proliferation of Ralstonia solanacearum OE1-1 in host intercellular spaces after the invasion of host plants is necessary for the virulence of this bacterium. A folate auxotroph, RM, in which a mini-Tn5 transposon was inserted into pabB encoding para-aminobenzoate synthase component I, lost its ability to vigorously proliferate in intercellular spaces along with its systemic infectivity and virulence after inoculation into roots and infiltration into leaves of tobacco plants. Complementation of RM with the pabB gene allowed the mutant to multiply in intercellular spaces and to cause disease. In tobacco plants that were pretreated with folate, RM was able to vigorously proliferate in the intercellular spaces and cause disease. Interestingly, when it was inoculated through cut stems, the mutant multiplied in the plants and was virulent. Moreover, the mutant multiplied well in stem fluids but not in intercellular fluids, suggesting that the folate concentration within intercellular spaces may be a limiting factor for bacterial proliferation. Therefore, folate biosynthesis contributes to the vigorous proliferation of bacteria in intercellular spaces and leads to systemic infectivity resulting in virulence.Bacterial wilt caused by Ralstonia solanacearum (29) is one of the most devastating bacterial plant diseases and causes severe losses in many important crops in the tropics, subtropics, and warm-temperature regions worldwide (16). R. solanacearum is a soilborne vascular bacterium. This bacterium generally invades plant roots via wounds or the natural openings from which secondary roots subsequently emerge (16,20). After invasion, bacteria first colonize and proliferate in intercellular spaces and then invade xylem vessels. Once bacteria have invaded the vessels, they multiply and travel rapidly throughout the entire plant. The plants then wilt when sap flow is reduced by the presence of large numbers of bacteria and the exopolysaccharide (EPS) slime that they produce (26).R. solanacearum OE1-1 is pathogenic for tobacco plants and induces necrotic lesions in tobacco leaves 60 h after infiltration (17,18). Although a compatible R. solanacearum strain, GMI1000, is pathogenic for tomato plants, similar to OE1-1, it is nonpathogenic to tobacco plants and elicits a hypersensitive response 24 h after infiltration of tobacco leaves, in contrast to OE1-1 (2,5,6,18,19,27). OE1-1 possesses hrp genes similar to those of R. solanacearum GMI1000 (2,18,19,27). These genes encode proteins which are parts of the type III secretion machinery, and their expression is regulated by HrpB (11,27). Type III secretion machinery-deficient mutants of OE1-1 lose the ability to colonize and proliferate in intercellular spaces, resulting in a loss of the ability to induce necrotic lesions and a lack of virulence (18). Therefore, type III effectors secreted through the type III secretion machinery of OE1-1 are involved in not only its virulence but also its induction of necrotic lesions in infiltrated tobacco leaves (18). Moreover, ...