Kazuhiro Nakaho

Distribution and multiplication of Ralstonia solanacearum in tomato plants with resistance derived from different origins

The distribution and multiplication of Ralstonia solanacearum in tomato plants of 11 resistant cultivars derived from different genetic sources and susceptible cultivar Ponderosa were examined. Bacterial multiplication in stems of resistant tomato plants was suppressed owing to the limitation of pathogen movement from the protoxylem or the primary xylem to other xylem tissues. The limitation was most conspicuous in Hawaii 7996. Grafting experiments indicated that the percentage of wilting of Ponderosa scions was less on Hawaii 7996 rootstocks than that on the most resistant rootstock (LS-89) used in Japan. Hawaii 7996 could be an alternative genetic source for breeding for resistance to bacterial wilt.

Beta-Cyanoalanine Synthase as a Molecular Marker for Induced Resistance by Fungal Glycoprotein Elicitor and Commercial Plant Activators

ABSTRACT The biocontrol agent Pythium oligandrum produces glycoprotein elicitor in the cell wall fraction, designated CWP, and induces resistance to a broad range of pathogens. To understand the mechanism of CWP-induced resistance to pathogens, gene expression at the early stage of CWP treatment in tomato roots was analyzed using a cDNA array. At 4 h after CWP treatment, 144 genes were up-regulated and 99 genes were down-regulated. In the 144 up-regulated genes, nine genes exhibited about eightfold increased expression. Analysis of the response of these nine genes to three commercial plant activators indicated that a high level of one gene, beta-cyanoalanine synthase gene (LeCAS) encoding hydrogen cyanide (HCN) detoxification enzyme, was stably induced in tomato roots by such treatment. However, expression of LeCAS was not significantly induced in tomato roots at 4 h by abiotic stresses, whereas only a very low level of induction of such expression by cold stress was observed. This LeCAS expression was also induced after exogenous treatment with a low level of 1-amino-cyclopropane-1-carboxylate as the precursor of ethylene, but not with either salicylic acid or methyl jas-monate. The induction of LeCAS expression in CWP-treated and plant activator-treated roots is likely to be caused by the detoxification of HCN during ethylene production. Transient activation of LeCAS expression caused by ethylene production in tomato roots may be a general phenomenon in fungal elicitor-induced and synthetic plant activator-induced resistance. LeCAS seems to be useful for screening possible novel plant activators for plant protection against pathogens.

Colonization of Pythium oligandrum in the tomato rhizosphere for biological control of bacterial wilt disease analyzed by real-time PCR and confocal laser-scanning microscopy.

It recently has been reported that the non-plant-pathogenic oomycete Pythium oligandrum suppresses bacterial wilt caused by Ralstonia solanacearum in tomato. As one approach to determine disease-suppressive mechanisms of action, we analyzed the colonization of P. oligandrum in rhizospheres of tomato using real-time polymerase chain reaction (PCR) and confocal laser-scanning microscopy. The real-time PCR specifically quantified P. oligandrum in the tomato rhizosphere that is reliable over a range of 0.1 pg to 1 ng of P. oligandrum DNA from 25 mg dry weight of soil. Rhizosphere populations of P. oligandrum from tomato grown for 3 weeks in both unsterilized and sterilized field soils similarly increased with the initial application of at least 5 x 10(5) oospores per plant. Confocal microscopic observation also showed that hyphal development was frequent on the root surface and some hyphae penetrated into root epidermis. However, rhizosphere population dynamics after transplanting into sterilized soil showed that the P. oligandrum population decreased with time after transplanting, particularly at the root tips, indicating that this biocontrol fungus is rhizosphere competent but does not actively spread along roots. Protection over the long term from root-infecting pathogens does not seem to involve direct competition. However, sparse rhizosphere colonization of P. oligandrum reduced the bacterial wilt as well as more extensive colonization, which did not reduce the rhizosphere population of R. solanacearum. These results suggest that competition for infection sites and nutrients in rhizosphere is not the primary biocontrol mechanism of bacterial wilt by P. oligandrum.

Transcriptome Analysis of Quantitative Resistance-Specific Response upon Ralstonia solanacearum Infection in Tomato

Bacterial wilt, caused by the soil-borne bacterium Ralstonia solanacearum, is a lethal disease of tomato, but the molecular mechanisms of the host resistance responses to R. solanacearum remain unclear. In this study, we report the first work describing the transcriptome of cultivar resistance and susceptible tomato cultivar after inoculation with R. solanacearum. To elucidate the characteristics of resistance early in the interaction, we analyzed microarrays for resistant cultivar LS-89 and susceptible cultivar Ponderosa 1 day after stem inoculation. No change in gene expression was detected for Ponderosa, but expression levels of over 140 genes, including pathogenesis-related, hormone signaling and lignin biosynthesis genes, increased in LS-89. Expression of β-1,3-glucanase genes increased substantially. In an immunohistochemical study, glucanase in LS-89 accumulated in the xylem and pith tissues surrounding xylem vessels filled with R. solanacearum. The expression of these genes also increased in four other resistant cultivars, but changed little in four susceptible cultivars in response to R. solanacearum, suggesting that similar reactions occur in other cultivars. These gene expression profiles will serve as fundamental information to elucidate the molecular mechanisms in the resistance response to R. solanacearum in tomato.

Enhanced defense responses in arabidopsis induced by the cell wall protein fractions from pythium oligandrum require SGT1, RAR1, NPR1 and JAR1

The cell wall protein fraction (CWP) is purified from the non-pathogenic biocontrol agent Pythium oligandrum and is composed of two glycoproteins (POD-1 and POD-2), which are structurally similar to class III elicitins. In tomato plants treated with CWP, jasmonic acid (JA)- and ethylene (ET)-dependent signaling pathways are activated, and resistance to Ralstonia solanaceraum is enhanced. To dissect CWP-induced defense mechanisms, we investigated defense gene expression and resistance to bacterial pathogens in Arabidopsis thaliana ecotype Col-0 treated with CWP. When the leaves of Col-0 were infiltrated with CWP, neither visible necrosis nor salicylic acid (SA)-responsive gene (PR-1 and PR-5) expression was induced. In contrast, JA-responsive gene (PDF1.2 and JR2) expression was up-regulated and the resistance to R. solanaceraum and Pseudomonas syringae pv. tomato DC3000 was enhanced in response to CWP. Such CWP-induced defense responses were completely compromised in CWP-treated coi1-1 and jar1-1 mutants with an impaired JA signaling pathway. The induction of defense-related gene expression after CWP treatment was partially compromised in ET-insensitive ein2-1 mutants, but not in SA signaling mutants or nahG transgenic plants. Global gene expression analysis using cDNA array also suggested that several other JA- and ET-responsive genes, but not SA-responsive genes, were up-regulated in response to CWP. Further analysis of CWP-induced defense responses using another eight mutants with impaired defense signaling pathways indicated that, interestingly, the induction of JA-responsive gene expression and enhanced resistance to two bacterial pathogens in response to CWP were completely compromised in rar1-1, rar1-21, sgt1a-1, sgt1b (edm1) and npr1-1 mutants. Thus, the CWP-induced defense system appears to be regulated by JA-mediated and SGT1-, RAR1- and NPR1-dependent signaling pathways.

Identification of Natural Diterpenes that Inhibit Bacterial Wilt Disease in Tobacco, Tomato and Arabidopsis

The soil-borne bacterial pathogen Ralstonia solanacearum invades a broad range of plants through their roots, resulting in wilting of the plant, but no effective protection against this disease has been developed. Two bacterial wilt disease-inhibiting compounds were biochemically isolated from tobacco and identified as sclareol and cis-abienol, labdane-type diterpenes. When exogenously applied to their roots, sclareol and cis-abienol inhibited wilt disease in tobacco, tomato and Arabidopsis plants without exhibiting any antibacterial activity. Microarray analysis identified many sclareol-responsive genes in Arabidopsis roots, including genes encoding or with a role in ATP-binding cassette (ABC) transporters, and biosynthesis and signaling of defense-related molecules and mitogen-activated protein kinase (MAPK) cascade components. Inhibition of wilt disease by sclareol was attenuated in Arabidopsis mutants defective in the ABC transporter AtPDR12, the MAPK MPK3, and ethylene and abscisic acid signaling pathways, and also in transgenic tobacco plants with reduced expression of NtPDR1, a tobacco homolog of AtPDR12. These results suggest that multiple host factors are involved in the inhibition of bacterial wilt disease by sclareol-related compounds.

Molecular typing of Japanese strains of Ralstonia solanacearum in relation to the ability to induce a hypersensitive reaction in tobacco

The genetic diversity of 120 Ralstonia solanacearum strains isolated from a variety of host plants across Japan was assessed on the basis of hypersensitive response (HR) in tobacco leaves and phylogenetic analyses of endoglucanase gene egl, hrpB, and gyrB. Phylogenetic analysis of egl revealed that only three strains belonged to phylotype IV, and 117 strains belonged to phylotype I. Partial sequences of HrpB were identical among phylotype I strains except for one strain. Analyses using the partial nucleotide sequences of the gyrB and egl gene fragments grouped phylotype I strains into 11 gyrB and 8 egl types, respectively, whereas analyses using the partial amino acid sequences of GyrB and Egl grouped phylotype I strains into 4 GyrB and 5 Egl types, respectively. Using multilocus sequence typing of GyrB and Egl, we identified 10 unique sequence types within the Japanese phylotype I strains. Strains belonging to the GyrB42 or GyrB66 type caused wilt in tobacco, and strains belonging to GyrB2 or GyrB9 type elicited HR, demonstrating that HR induction in tobacco is genetically differentiated in the Japanese strains of R. solanacearum.

Temperature-upshift-mediated revival from the sodium-pyruvate-recoverable viable but nonculturable state induced by low temperature in Ralstonia solanacearum: linear regression analysis.

The viable but nonculturable (VBNC) state is induced in stress-injured bacterial cells. VBNC cells lose their ability to grow on standard media, but some of the cells can recover on media supplemented with H2O2-degrading compounds such as sodium pyruvate (SP). Here, VBNC cells that can recover on such media are called SP-recoverable VBNC cells; VBNC cells that do not recover are called SP-unrecoverable VBNC cells. On the basis of previous findings, we hypothesized that cells of Ralstonia solanacearum in which low temperature had induced the SP-recoverable VBNC state would regain their ability to grow on standard solid media after exposure to moderate temperature. To test this, cell suspensions of R. solanacearum were incubated at 5°C. When SP-recoverable VBNC cells were the only culturable cells present, the cell suspensions were exposed to 25°C. The temperature upshift caused an initial rapid increase in the number of active cells able to grow on standard solid media, followed by a more gradual increase. Linear regression analyses suggested that this increase was not attributable to the regrowth of a low level of residual active cells. In addition, all the revived cells tested caused wilt symptoms in susceptible tomato plants. When SP-unrecoverable VBNC cells were the only viable cells present in the 5°C microcosms, the cell suspensions were exposed to 25°C. In this case, no culturable cells were detected. Therefore, these data strongly suggest that the SP-recoverable VBNC cells regained their ability to grow on standard solid media after the temperature upshift and that the revived cells were virulent.

Transcriptional profile of tomato roots exhibiting Bacillus thuringiensis-induced resistance to Ralstonia solanacearum.

Key messageActivation of SA-dependent signaling pathway and suppression of JA-dependent signaling pathway seem to play key roles inB. thuringiensis-induced resistance toR. solanacearumin tomato plants.AbstractBacillus thuringiensis, a well-known and effective bio-insecticide, has attracted considerable attention as a potential biological control agent for the suppression of plant diseases. Treatment of tomato roots with a filter-sterilized cell-free filtrate (CF) of B. thuringiensis systemically suppresses bacterial wilt caused by Ralstonia solanacearum through systemic activation of the plant defense system. Comparative analysis of the expression of the Pathogenesis-Related 1(P6) gene, a marker for induced resistance to pathogens, in various tissues of tomato plants treated with CF on their roots suggested that the B. thuringiensis-induced defense system was activated in the leaf, stem, and main root tissues, but not in the lateral root tissue. At the same time, the growth of R. solanacearum was significantly suppressed in the CF-treated main roots but not in the CF-treated lateral roots. This distinct activation of the defense reaction and suppression of R. solanacearum were reflected by the differences in the transcriptional profiles of the main and lateral tissues in response to the CF. In CF-treated main roots, but not CF-treated lateral roots, the expression of several salicylic acid (SA)-responsive defense-related genes was specifically induced, whereas jasmonic acid (JA)-related gene expression was either down-regulated or not induced in response to the CF. On the other hand, genes encoding ethylene (ET)-related proteins were induced equally in both the main and lateral root tissues. Taken together, the co-activation of SA-dependent signaling pathway with ET-dependent signaling pathway and suppression of JA-dependent signaling pathway may play key roles in B. thuringiensis-induced resistance to R. solanacearum in tomato.

Contribution of the type II secretion system in systemic infectivity of Ralstonia solanacearum through xylem vessels

Ralstonia solanacearum strain OE1-1 (OE1-1) systemically invades tobacco plants and causes bacterial wilt. A type II secretion system (T2SS)-deficient mutant of OE1-1, derived from EZ::TNtransposon-insertion, retained the ability of the parent strain to produce exopolysaccharide in vitro and grow in intercellular spaces immediately after invasion of host plants, but lost the ability to systemically infect the host. With transmission electron microscopy, the mutant was not observed in xylem vessels. These findings suggest that the T2SS contributes to systemic infection by enabling the bacteria to invade xylem vessels.