Is the High Basal Level of Salicylic Acid Important for Disease Resistance in Potato?

Potato (Solanum tuberosum) plants contain a high basal level of salicylic acid (SA), the role of which in disease resistance is currently unclear. Here we report that, in spite of a drastic reduction in total SA levels in transgenic potato plants expressing the bacterial salicylate hydroxylase gene (nahG), there was no significant increase in disease severity when infected by Phytophthora infestans. Therefore, the high basal level of SA does not lead to constitutive resistance in healthy potato plants. However, in contrast to control plants, arachidonic acid failed to induce systematic acquired resistance (SAR) in nahG plants against P. infestans, indicating an essential role of SA in potato SAR. These results suggest that in potato the development of SAR against P. infestans may involve increased sensitivity of the plant to SA.     

Abnormal callose response phenotype and hypersusceptibility to Peronospoara parasitica in defence-compromised arabidopsis nim1-1 and salicylate hydroxylase-expressing plants

To investigate the impact of induced host defenses on the virulence of a compatible Peronospora parasitica strain on Arabidopsis thaliana, we examined growth and development of this pathogen in nim1-1 mutants and transgenic salicylate hydroxylase plants. These plants are unable to respond to or accumulate salicylic acid (SA), respectively, are defective in expression of systemic acquired resistance (SAR), and permit partial growth of some normally avirulent pathogens. We dissected the P. parasitica life cycle into nine stages and compared its progression through these stages in the defense-compromised hosts and in wild-type plants. NahG plants supported the greatest accumulation of pathogen biomass and conidiophore production, followed by nim1-1 and then wild-type plants. Unlike the wild type, NahG and nim1-1 plants showed little induction of the SAR gene PR-1 after colonization with P parasitica, which is similar to our previous observations. We examined the frequency and morphology of callose deposits around parasite haustoria and found significant differences between the three hosts. NahG plants showed a lower fraction of haustoria surrounded by thick callose encasements and a much higher fraction of haustoria with callose limited to thin collars around haustorial necks compared to wild type, whereas nim1-1 plants were intermediate between NahG and wild type. Chemical induction of SAR in plants colonized by P. parasitica converted the extrahaustorial callose phenotype in NahG to resemble closely the wild-type pattern, but had no effect on nim1-1 plants. These results suggest that extrahaustorial callose deposition is influenced by the presence or lack of SA and that this response may be sensitive to the NIM1/NPR1 pathway. Additionally, the enhanced susceptibility displayed by nim1-1 and NahG plants shows that even wild-type susceptible hosts exert defense functions that reduce disease severity and pathogen fitness.     

A two-strain mixture of rhizobacteria elicits induction of systemic resistance against Pseudomonas syringae and Cucumber mosaic virus coupled to promotion of plant growth on Arabidopsis thaliana

We evaluated a commercial biopreparation of plant growth-promoting rhizobacteria (PGPR) strains Bacillus subtilis GB03 and B. amyloliquefaciens IN937a formulated with the carrier chitosan (BioYield) for its capacity to elicit growth promotion and induced systemic resistance against infection by Cucumber Mosaic Virus (CMV) and Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana. The biopreparation promoted plant growth of Arabidopsis hormonal mutants, which included auxin, gibberellic acid, ethylene, jasmonate, salicylic acid, and brassinosteroid insensitive lines as well as each wild-type. The biopreparation protected plants against CMV based on disease severity in wild-type plants. However, virus titre was not lower in control plants and those treated with biopreparation, suggesting that the biopreparation induced tolerance rather than resistance against CMV. Interestingly, the biopreparation induced resistance against CMV in NahG plants, as evidenced by both reduced disease severity and virus titer. The biopreparation also elicited induced resistance against P. syringae pv. tomato in the wild-type but not in NahG transgenic plants, which degrade endogenous salicylic acid, indicating the involvement of salicylic acid signaling. Our results indicate that some PGPR strains can elicit plant growth promotion by mechanisms that are different from known hormonal signaling pathways. In addition, the mechanism for elicitation of induced resistance by PGPR may be pathogen-dependent. Collectively, the two-Bacilli strain mixture can be utilized as a biological inoculant for both protection of plant against bacterial and viral pathogens and enhancement of plant growth.     

Partial resistance of tomato to Phytophthora infestans is not dependent upon ethylene,jasmonic acid,or salicylic acid signaling pathways

We compared tomato defense responses to Phytophthora infestans in highly compatible and partially compatible interactions. The highly compatible phenotype was achieved with a tomato-specialized isolate of P. infestans, whereas the partially compatible phenotype was achieved with a nonspecialized isolate. As expected, there was induction of the hypersensitive response (HR) earlier during the partially compatible interaction. However, contrary to our expectation, pathogenesis-related (PR) gene expression was not stimulated sooner in the partially compatible interaction. While the level of PR gene expression was quite similar in the two interactions, the LeDES gene (which encodes an enzyme necessary for the production of divinyl ethers) was expressed at a much higher level in the partially compatible interaction at 48 h after inoculation. Host reaction to the different pathogen genotypes was not altered (compared with wild type) in mutant tomatoes that were ethylene-insensitive (Never-ripe) or those with reduced ability to accumulate jasmonic acid (def-1). Similarly, host reaction was not altered in NahG transgenic tomatoes unable to accumulate salicylic acid. These combined data indicate that partial resistance in tomato to P. infestans is independent of ethylene, jasmonic acid, and salicylic acid signaling pathways.     

Expression of tobacco class II catalase gene activates the endogenous homologous gene and is associated with disease resistance in transgenic potato plants

We have previously shown that healthy potato plants respond poorly to salicylic acid (SA) for activating disease resistance against the late blight fungal pathogen Phytophthora infestans. However, SA is essential for the establishment of potato systemic acquired resistance (SAR) against P. infestans after treatment with the fungal elicitor arachidonic acid (AA). To understand the molecular mechanisms through which AA induces SA-dependent SAR in potato, we have recently studied the expression of potato class II catalase (Cat2St) in comparison with its tobacco homologue, Cat2Nt, which has previously been shown to bind SA. In the present study, we show that tobacco Cat2Nt is expressed at high levels and accounts for almost half of total SA-binding activity detected in tobacco leaves. In contrast, potato Cat2St is not expressed in healthy leaves, which is associated with the low SA responsiveness of potato plants for activation of disease resistance mechanisms. Upon treatment with AA, expression of potato Cat2St is induced not only in AA-treated leaves, but also in the upper untreated parts of the plants, concomitant with the establishment of SA -dependent SAR to P. infestans. Moreover, expression of the tobacco Cat2Nt gene in transgenic potato plants leads to constitutive expression of the endogenous potato Cat2St gene and is associated with enhanced resistance to P. infestans. These results collectively indicate that plant SA-binding class II catalases may play an important role in the development of disease resistance, possibly by serving as biological targets of SA.     

StCDPK5 confers resistance to late blight pathogen but increases susceptibility to early blight pathogen in potato via reactive oxygen species burst

? Potato (Solanum tuberosum) calcium-dependent protein kinase (StCDPK5) has been shown to phosphorylate the N-terminal region of plasma membrane RBOH (respiratory burst oxidase homolog) proteins, and participate in StRBOHB-mediated reactive oxygen species (ROS) burst. The constitutively active form, StCDPK5VK, provides a useful tool for gain-of-function analysis of RBOH in defense responses. ? StCDPK5- and StCDPK5VK-green fluorescent protein fusion proteins were predominantly targeted to the plasma membrane, and conditional expression of StCDPK5VK activated StRBOHA-D. The interaction was confirmed by bimolecular fluorescence complementation assay. We generated transgenic potato plants containing StCDPK5VK under the control of a pathogen-inducible promoter to investigate the role of ROS burst on defense responses to blight pathogens. ? Virulent isolates of the late blight pathogen Phytophthora infestans and the early blight pathogen Alternaria solani induced hypersensitive response-like cell death accompanied by ROS production at the infection sites of transgenic plants. Transgenic plants showed resistance to the near-obligate hemibiotrophic pathogen P.?infestans and, by contrast, increased susceptibility to the necrotrophic pathogen A.?solani. ? These results indicate that RBOH-dependent ROS contribute to basal defense against near-obligate pathogens, but have a negative role in resistance or have a positive role in expansion of disease lesions caused by necrotrophic pathogens.     

Probenazole induces systemic acquired resistance in Arabidopsis with a novel type of action

Probenazole (PBZ; 3-allyloxy-1,2-benzisothiazole-1,1-dioxide), which is the active ingredient in Oryzemate, has been used widely in Asia to protect rice plants against the rice blast fungus Magnaporthe grisea. To study PBZ's mode of action, we analyzed its ability, as well as that of its active metabolite 1, 2-benzisothiazol-3 (2H)-one 1,1-dioxide (BIT) to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild-type Arabidopsis treated with PBZ or BIT exhibited increased expression of several pathogenesis-related genes, increased levels of total salicylic acid (SA), and enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC 3000 and the oomycete pathogen Peronospora parasitica Emco5. The role of several defense signaling hormones, such as SA, ethylene and jasmonic acid (JA), in activating resistance following PBZ or BIT treatment was analyzed using NahG transgenic plants and etr1-1 and coi1-1 mutant plants, respectively. In addition, the involvement of NPR1, a key component in the SA signaling pathway leading to defense responses, was assessed. PBZ or BIT treatment did not induce disease resistance or PR-1 expression in NahG transgenic or npr1 mutant plants, but it did activate these phenomena in etr1-1 and coi 1-1 mutant plants. Thus SA and NPR1 appear to be required for PBZ- and BIT-mediated activation of defense responses, while ethylene and JA are not. Furthermore, our data suggest that PBZ and BIT comprise a novel class of defense activators that stimulate the SA/NPR1-mediated defense signaling pathway upstream of SA.     

Quantitative resistance of potato to Pectobacterium atrosepticum and Phytophthora infestans: integrating PAMP-triggered response and pathogen growth

While the mechanisms underlying quantitative resistance of plants to pathogens are still not fully elucidated, the Pathogen-Associated Molecular Patterns (PAMPs)-triggered response model suggests that such resistance depends on a dynamic interplay between the plant and the pathogen. In this model, the pathogens themselves or elicitors they produce would induce general defense pathways, which in turn limit pathogen growth and host colonisation. It therefore suggests that quantitative resistance is directly linked to a common set of general host defense mechanisms, but experimental evidence is still inconclusive. We tested the PAMP-triggered model using two pathogens (Pectobacterium atrosepticum and Phytophthora infestans) differing by their infectious processes and five potato cultivars spanning a range of resistance levels to each pathogen. Phenylalanine ammonia-lyase (PAL) activity, used as a defense marker, and accumulation of phenolics were measured in tuber slices challenged with lipopolysaccharides from P. atrosepticum or a concentrated culture filtrate from P. infestans. PAL activity increased following treatment with the filtrate but not with lipopolysaccharides, and varied among cultivars. It was positively related to tuber resistance to P. atrosepticum, but negatively related to tuber resistance to P. infestans. It was also positively related to the accumulation of total phenolics. Chlorogenic acid, the main phenolic accumulated, inhibited growth of both pathogens in vitro, showing that PAL induction caused active defense against each of them. Tuber slices in which PAL activity had been induced before inoculation showed increased resistance to P. atrosepticum, but not to P. infestans. Our results show that inducing a general defense mechanism does not necessarily result in quantitative resistance. As such, they invalidate the hypothesis that the PAMP-triggered model alone can explain quantitative resistance. We thus designed a more complex model integrating physiological host response and a key pathogen life history trait, pathogen growth, to explain the differences between the two pathosystems.     

Induced parasitoid attraction by Arabidopsis thaliana: involvement of the octadecanoid and the salicylic acid pathway

Plants can use indirect defence mechanisms to protect themselves against herbivorous insects. An example of such an indirect defence mechanism is the emission of volatiles by plants induced by herbivore feeding. These volatiles can attract the natural enemies of these herbivores, for example, parasitoid wasps. Here, it is shown that the octadecanoid and the salicylic acid pathways are involved in the induced attraction of the parasitoid wasp Cotesia rubecula by Arabidopsis thaliana infested with the herbivore Pieris rapae. Besides exogenous application of jasmonic acid or salicylic acid, use is also made of transgenic Arabidopsis that do not show induced jasmonic acid levels after wounding (S-12) and transgenic Arabidopsis that do not accumulate salicylic acid (NahG). Treatment of Arabidopsis with jasmonic acid resulted in an increased attraction of parasitoid wasps compared with untreated plants, whereas treatment with salicylic acid did not. Transgenic plants impaired in the octadecanoid or the salicylic acid pathway were less attractive than wild-type plants.     

Down-regulation of antioxidative capacity in a transgenic tobacco which fails to develop acquired resistance to necrotization caused by TMV

Antioxidant status was assayed in leaves of two local lesion hosts of tobacco mosaic virus (TMV), namely in wild-type Xanthi-nc tobacco and in NahG transgenic tobacco, the latter of which is not able to accumulate salicylic acid (SA) and therefore is unable to develop systemic acquired resistance (SAR). Activities of several enzymes related to antioxidative defense, and the levels of glutathione, chlorogenic acid and rutin were studied. The majority of antioxidant enzymes were less active in uninfected NahG tobacco than in Xanthi-nc. Furthermore, important enzymatic and non-enzymatic antioxidants were down-regulated in TMV-infected NahG plants, as compared to Xanthi-nc. Correspondingly, SA pretreatment primed the leaves for stronger induction of antioxidants in infected Xanthi-nc, but not in NahG tobaccos. The antioxidant status of NahG tobacco even decreased after an attempted induction of SAR, while the antioxidative level increased in Xanthi-nc leaves in which the SAR was successfully induced. After infection, a greater accumulation of superoxide and H 2 O 2 , and a more intensive necrotization was positively correlated with the reduced capability of NahG leaf tissue to detoxify reactive oxygen species.     

Disease resistance conferred by expression of a gene encoding H2O2-generating glucose oxidase in transgenic potato plants.

Plant defense responses to pathogen infection involve the production of active oxygen species, including hydrogen peroxide (H2O2). We obtained transgenic potato plants expressing a fungal gene encoding glucose oxidase, which generates H2O2 when glucose is oxidized. H2O2 levels were elevated in both leaf and tuber tissues of these plants. Transgenic potato tubers exhibited strong resistance to a bacterial soft rot disease caused by Erwinia carotovora subsp carotovora, and disease resistance was sustained under both aerobic and anaerobic conditions of bacterial infection. This resistance to soft rot was apparently mediated by elevated levels of H2O2, because the resistance could be counteracted by exogenously added H2O2-degrading catalase. The transgenic plants with increased levels of H2O2 also exhibited enhanced resistance to potato late blight caused by Phytophthora infestans. The development of lesions resulting from infection by P. infestans was significantly delayed in leaves of these plants. Thus, the expression of an active oxygen species-generating enzyme in transgenic plants represents a novel approach for engineering broad-spectrum disease resistance in plants.     

Brassinosteroid functions in a broad range of disease resistance in tobacco and rice

Brassinolide (BL), considered to be the most important brassinosteroid (BR) and playing pivotal roles in the hormonal regulation of plant growth and development, was found to induce disease resistance in plants. To study the potentialities of BL activity on stress responding systems, we analyzed its ability to induce disease resistance in tobacco and rice plants. Wild-type tobacco treated with BL exhibited enhanced resistance to the viral pathogen tobacco mosaic virus (TMV), the bacterial pathogen Pseudomonas syringae pv. tabaci (Pst), and the fungal pathogen Oidium sp. The measurement of salicylic acid (SA) in wild-type plants treated with BL and the pathogen infection assays using NahG transgenic plants indicate that BL-induced resistance does not require SA biosynthesis. BL treatment did not induce either acidic or basic pathogenesis-related (PR) gene expression, suggesting that BL-induced resistance is distinct from systemic acquired resistance (SAR) and wound-inducible disease resistance. Analysis using brassinazole 2001, a specific inhibitor for BR biosynthesis, and the measurement of BRs in TMV-infected tobacco leaves indicate that steroid hormone-mediated disease resistance (BDR) plays part in defense response in tobacco. Simultaneous activation of SAR and BDR by SAR inducers and BL, respectively, exhibited additive protective effects against TMV and Pst, indicating that there is no cross-talk between SAR- and BDR-signaling pathway downstream of BL. In addition to the enhanced resistance to a broad range of diseases in tobacco, BL induced resistance in rice to rice blast and bacterial blight diseases caused by Magnaporthe grisea and Xanthomonas oryzae pv. oryzae, respectively. Our data suggest that BDR functions in the innate immunity system of higher plants including dicotyledonous and monocotyledonous species.     

Effects of endogenous salicylic acid on nodulation in the model legumes Lotus japonicus and Medicago truncatula

The exogenous addition of salicylic acid (SA) was previously shown to inhibit indeterminate but not determinate-type nodulation. We sought to extend these results by modulating endogenous levels of SA through the transgenic expression of salicylate hydroxylase (NahG) in both stably transformed Lotus japonicus and composite Medicago truncatula plants. NahG expression in L. japonicus resulted in a marked reduction of SA levels. This reduction correlated with an increase in the number of infections and mean nodule number when compared to controls. However, a complicating factor was that NahG-expressing plants had greater root growth. Spot inoculations of NahG-expressing L. japonicus plants confirmed increased nodulation in these plants. Consistent with the reported inhibitory effects of exogenous SA on indeterminate-type nodulation, NahG expression in M. truncatula plants led to enhanced nodulation and infection. These data point to an important role for SA-mediated plant defense pathways in controlling nodule formation on both determinate and indeterminate nodule-forming hosts.     

Down-regulation of Antioxidative Capacity in a Transgenic Tobacco which Fails to Develop Acquired Resistance to Necrotization Caused by TMV

Antioxidant status was assayed in leaves of two local lesion hosts of tobacco mosaic virus (TMV), namely in wild-type Xanthi-nc tobacco and in NahG transgenic tobacco, the latter of which is not able to accumulate salicylic acid (SA) and therefore is unable to develop systemic acquired resistance (SAR). Activities of several enzymes related to antioxidative defense, and the levels of glutathione, chlorogenic acid and rutin were studied. The majority of antioxidant enzymes were less active in uninfected NahG tobacco than in Xanthi-nc. Furthermore, important enzymatic and non-enzymatic antioxidants were down-regulated in TMV-infected NahG plants, as compared to Xanthi-nc. Correspondingly, SA pretreatment primed the leaves for stronger induction of antioxidants in infected Xanthi-nc, but not in NahG tobaccos. The antioxidant status of NahG tobacco even decreased after an attempted induction of SAR, while the antioxidative level increased in Xanthi-nc leaves in which the SAR was successfully induced. After infection, a greater accumulation of superoxide and H 2 O 2, and a more intensive necrotization was positively correlated with the reduced capability of NahG leaf tissue to detoxify reactive oxygen species.