Rewiring mitogen-activated protein kinase cascade by positive feedback confers potato blight resistance

Late blight, caused by the notorious pathogen Phytophthora infestans, is a devastating disease of potato (Solanum tuberosum) and tomato (Solanum lycopersicum), and during the 1840s caused the Irish potato famine and over one million fatalities. Currently, grown potato cultivars lack adequate blight tolerance. Earlier cultivars bred for resistance used disease resistance genes that confer immunity only to some strains of the pathogen harboring corresponding avirulence gene. Specific resistance gene-mediated immunity and chemical controls are rapidly overcome in the field when new pathogen races arise through mutation, recombination, or migration from elsewhere. A mitogen-activated protein kinase (MAPK) cascade plays a pivotal role in plant innate immunity. Here we show that the transgenic potato plants that carry a constitutively active form of MAPK kinase driven by a pathogen-inducible promoter of potato showed high resistance to early blight pathogen Alternaria solani as well as P. infestans. The pathogen attack provoked defense-related MAPK activation followed by induction of NADPH oxidase gene expression, which is implicated in reactive oxygen species production, and resulted in hypersensitive response-like phenotype. We propose that enhancing disease resistance through altered regulation of plant defense mechanisms should be more durable and publicly acceptable than engineering overexpression of antimicrobial proteins.     

Role of hydrogen peroxide and ascorbate-glutathione pathway in host resistance to bacterial wilt of eggplant

Ralstonia solanacearum, a soil-borne bacterium causes bacterial wilt, is a lethal disease of eggplant (Solanum melongena L.). However, the first line of defense mechanism of R. solanacearum infection remains unclear. The present study focused on the role of induced H₂O₂, defense-related enzymes of ascorbate-glutathione pathway variations in resistant and susceptible cultivars of eggplant under biotic stress. Fifteen cultivars of eggplant were screened for bacterial wilt resistance, and the concentration of antioxidant enzymes were estimated upon infection with R. solanacearum. A quantitative real-time PCR was also carried out to study the expression of defense genes. The concentration of H₂O₂ in the pathogen inoculated seedlings was two folds higher at 12 h after pathogen inoculation compared to control. Antioxidant enzymes of ascorbate-glutathione pathway were rapidly increased in resistant cultivars followed by susceptible and highly susceptible cultivars upon pathogen inoculation. The enzyme activity of ascorbate-glutathione pathway correlates by amplification of their defense genes along with pathogenesis-related protein-1a (PR-1a). The expressions of defense genes increased 2.5?3.5 folds in resistant eggplant cultivars after pathogen inoculation. The biochemical and molecular markers provided an insight to understand the first line of defense responses in eggplant cultivars upon inoculation with the pathogen.     

Regulatory role of nitric oxide in lipopolysaccharides-triggered plant innate immunity

Recent studies have suggested that lipopolysaccharides (LPS) induce nitric oxide (NO) production and defense gene expression in plants. Our current work investigated the signaling mechanism of NO and the role of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) in LPS-induced innate immunity of Arabidopsis (Arabidopsis thaliana). We have provided evidence that LPS-elicited NO generation as well as increased antioxidant enzyme activities capable of maintaining the redox state could be important to protect plants against oxidative damage from pathogen attack. In addition, LPS-activated defense responses, including callose deposition and defense-related gene expression, are regulated through an NPR1-dependent signaling pathway. Our results contribute to elucidation of the signaling mechanism of NO and highlight an important role of NPR1 in modulating LPS-triggered innate immunity in plants. However, further research is necessary to clarify the cross-talk between mitochondria and NO on activating LPS-induced defense responses, and the regulatory mechanism of NO in LPS-induced innate immunity needs further improvement.     

Wheat defense genes in fungal (Puccinia striiformis) infection

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most important diseases of wheat worldwide. To isolate defense-related genes against the pathogen, a suppression subtractive hybridization library was constructed for an incompatible interaction. From the library, 652 sequences were determined to be unigenes, of which 31 were determined as genes involved in signal transduction and 77 were predicted to encode defense-related proteins. Expression patterns of 12 selected signal transduction and defense-related genes were determined using quantitative real-time polymerase chain reaction. Signal transduction genes started increasing their expression at 12 h post inoculation (hpi), and expressions of the most of the transport and resistance-related genes were induced at 18 hpi. The gene expression results indicate specific molecular and cellular activities during the incompatible interaction between wheat and the stripe rust pathogen. In general, the expression increase of wheat signal transduction genes soon after inoculation with the pathogen inducing various defense-related genes, including reactive oxygen species, ATP-binding cassette (ABC) transporters, pathogenesis-related proteins, and genes involved in the phenylpropanoid pathway. The activities of these defense genes work in a sequential and concerted manner to result in a hypersensitive response.     

Induction of Defense-Related Responses in Cf9 Tomato Cells by the AVR9 Elicitor Peptide of Cladosporium fulvum Is Developmentally Regulated

The AVR9 elicitor from the fungal pathogen Cladosporium fulvum induces defense-related responses, including cell death, specifically in tomato (Lycopersicon esculentum Mill.) plants that carry the Cf-9 resistance gene. To study biochemical mechanisms of resistance in detail, suspension cultures of tomato cells that carry the Cf-9 resistance gene were initiated. Treatment of cells with various elicitors, except AVR9, induced an oxidative burst, ion fluxes, and expression of defense-related genes. Agrobacterium tumefaciens-mediated transformation of Cf9 tomato leaf discs with Avr9-containing constructs resulted efficiently in transgenic callus formation. Although transgenic callus tissue showed normal regeneration capacity, transgenic plants expressing both the Cf-9 and the Avr9 genes were never obtained. Transgenic F₁ seedlings that were generated from crosses between tomato plants expressing the Avr9 gene and wild-type Cf9 plants died within a few weeks. However, callus cultures that were initiated on cotyledons from these seedlings could be maintained for at least 3 months and developed similarly to callus cultures that contained only the Cf-9 or the Avr9 gene. It is concluded, therefore, that induction of defense responses in Cf9 tomato cells by the AVR9 elicitor is developmentally regulated and is absent in callus tissue and cell-suspension cultures, which consists of undifferentiated cells. These results are significant for the use of suspension-cultured cells to investigate signal transduction cascades.     

Proteomic analysis of the compatible interaction between Vitis vinifera and Plasmopara viticola

We analyzed the proteome of grapevine (Vitis vinifera) leaves 24, 48 and 96 h post infection (hpi) with the downy mildew pathogen Plasmopara viticola. Total proteins were separated on 2-DE gels. By MS analysis, we identified 82 unique grapevine proteins differentially expressed after infection. Upregulated proteins were often included in the functional categories of general metabolism and stress response, while proteins related to photosynthesis and energy production were mostly downregulated. As expected, the activation of a defense reaction was observed more often at the late time point, consistent with the establishment of a compatible interaction. Most proteins involved in resistance were isoforms of different PR-10 pathogenesis-related proteins. Although > 50 differentially expressed protein isoforms were observed at 24 and 96 hpi, only 18 were detected at 48 hpi and no defense-related proteins were among this group. This profile suggests a transient breakdown in defense responses accompanying the onset of disease, further supported by gene expression analyses and by a western blot analysis of a PR-10 protein. Our data reveal the complex modulation of plant metabolism and defense responses during compatible interactions, and provide insight into the underlying molecular processes which may eventually yield novel strategies for pathogen control in the field.     

Glycolate oxidase modulates reactive oxygen species-mediated signal transduction during nonhost resistance in Nicotiana benthamiana and Arabidopsis

In contrast to gene-for-gene disease resistance, nonhost resistance governs defense responses to a broad range of potential pathogen species. To identify specific genes involved in the signal transduction cascade associated with nonhost disease resistance, we used a virus-induced gene-silencing screen in Nicotiana benthamiana, and identified the peroxisomal enzyme glycolate oxidase (GOX) as an essential component of nonhost resistance. GOX-silenced N. benthamiana and Arabidopsis thaliana GOX T-DNA insertion mutants are compromised for nonhost resistance. Moreover, Arabidopsis gox mutants have lower H(2)O(2) accumulation, reduced callose deposition, and reduced electrolyte leakage upon inoculation with hypersensitive response-causing nonhost pathogens. Arabidopsis gox mutants were not affected in NADPH oxidase activity, and silencing of a gene encoding NADPH oxidase (Respiratory burst oxidase homolog) in the gox mutants did not further increase susceptibility to nonhost pathogens, suggesting that GOX functions independently from NADPH oxidase. In the two gox mutants examined (haox2 and gox3), the expression of several defense-related genes upon nonhost pathogen inoculation was decreased compared with wild-type plants. Here we show that GOX is an alternative source for the production of H(2)O(2) during both gene-for-gene and nonhost resistance responses.     

Proteome-level investigation of Cucumis sativus-derived resistance to Sphaerotheca fuliginea

Powdery mildew, caused by Sphaerotheca fuliginea (Sf), is a widely distributed and destructive disease of greenhouse and field-grown cucumber plants and causes great yield loss. The objective of this research is to tentatively identify proteins that are differentially expressed in cucumber and are involved in modulating resistance to Sf-inoculation. We comparatively analyzed proteins differentially expressed in Sf-inoculated cucumber leaves using a pair of sister lines, B21-a-2-2-2 (highly susceptible) and B21-a-2-1-2 (highly resistant). To eliminate the interference of ribulose-1,5-bisphosphate carboxylase with low-abundance proteins, total proteins were pre-fractionated by 24 % polyethylene glycol (PEG) and the proteins from supernatant were analyzed by 2-DE. We were successful in establishing the identities of 20 proteins and those identified from the resistant line included proteins involved in metabolic, regulatory, and defense pathways. Our findings are discussed within the context of C. sativus–S. fuliginea interaction and tolerance to this pathogen. The results suggest that the resistance in cucumber is closely related to the enhancement of its primary metabolism, and ethylene modulated signaling in cucumber defense responses against powdery mildew, then defense-related proteins can be up-regulated as a result of altered gene expression.     

Jumonji C Domain Protein JMJ705-Mediated Removal of Histone H3 Lysine 27 Trimethylation Is Involved in Defense-Related Gene Activation in Rice

Histone methylation is an important epigenetic modification in chromatin function, genome activity, and gene regulation. Dimethylated or trimethylated histone H3 lysine 27 (H3K27me2/3) marks silent or repressed genes involved in developmental processes and stress responses in plants. However, the role and the mechanism of the dynamic removal of H3K27me2/3 during gene activation remain unclear. Here, we show that the rice (Oryza sativa) Jumonji C (jmjC) protein gene JMJ705 encodes a histone lysine demethylase that specifically reverses H3K27me2/3. The expression of JMJ705 is induced by stress signals and during pathogen infection. Overexpression of the gene reduces the resting level of H3K27me2/3 resulting in preferential activation of H3K27me3-marked biotic stress-responsive genes and enhances rice resistance to the bacterial blight disease pathogen Xanthomonas oryzae pathovar oryzae. Mutation of the gene reduces plant resistance to the pathogen. Further analysis revealed that JMJ705 is involved in methyl jasmonate–induced dynamic removal of H3K27me3 and gene activation. The results suggest that JMJ705 is a biotic stress-responsive H3K27me2/3 demethylase that may remove H3K27me3 from marked defense-related genes and increase their basal and induced expression during pathogen infection.     

Pathogen recognition and signal transduction by the Pto kinase

In tomato, the disease resistance genePto confers resistance to bacterial speck disease by recognizing the expression of a corresponding avirulence gene,avrPto, in the pathogenPseudomonas syringae pv.tomato (Martinet al. 1993). Similar “gene-for-gene” interactions occur in many plant-pathogen associations (Flor 1971). Such recognition events often lead to the activation in the plant of a variety of defense responses including a rapid induction of localized necrosis at the site of infection (the hypersensitive response, HR), increased expression of defense-related genes, production of antimicrobial compounds, lignin formation, and the oxidative burst (Lambet al. 1989, Mehdy 1994). As a result, the pathogen is contained at the infection site and its growth is inhibited.Pto encodes a serine/threonine protein kinase and belongs to a clustered multigene family. Another member of thePto family calledFen confers no known disease resistance, but mediates a hypersensitive-like reaction in the plant to the insecticide fenthion (Martinet al. 1994). We are interested in a number of fundamental questions concerning the Pto signaling pathways. What is the molecular basis of thePto-avrPto gene-for-gene interaction? What are the components involved in thePto-mediated signal transduction chain? How does thePto kinase activate complex defense responses? This paper summarizes our recent progress towards understanding these questions.     

Identification of rice genes induced in a rice blast-resistant mutant

To clarify mechanisms of rice blast resistance in rice plants we used suppression subtractive hybridization (SSH) to isolate genes induced upon rice blast inoculation in a rice blast-resistant mutant. A total of 26 rice cDNAs were isolated and found to have elevated expression upon rice blast infection in a rice blast-resistant derivative, SHM-11, of the rice cultivar, Sanghaehyanghyella. Sequencing of the cDNAs revealed that many of the proteins they encoded had been previously described as involved in plant responses against pathogen attack. Two interesting groups of the defense-related proteins consisted of three different PR5 homologues and four different protease inhibitors, all highly expressed in the rice blast mutant. Genes encoding proteins involved in signal transduction and regulation were also identified, including translation initiation factor eIF5A, C2 domain DNA binding protein, putative rice EDS and putative receptor like kinase. Most of the identified cDNAs were highly expressed 24 h after blast inoculation. Our results suggest that a pathway regulating defense gene expression may be altered in the mutant, resulting in early induction of the defense genes upon fungal infection.     

Temperature Modulates Plant Defense Responses through NB-LRR Proteins

An elevated growth temperature often inhibits plant defense responses and renders plants more susceptible to pathogens. However, the molecular mechanisms underlying this modulation are unknown. To genetically dissect this regulation, we isolated mutants that retain disease resistance at a higher growth temperature in Arabidopsis. One such heat-stable mutant results from a point mutation in SNC1, a NB-LRR encoding gene similar to disease resistance (R) genes. Similar mutations introduced into a tobacco R gene, N, confer defense responses at elevated temperature. Thus R genes or R-like genes involved in recognition of pathogen effectors are likely the causal temperature-sensitive component in defense responses. This is further supported by snc1 intragenic suppressors that regained temperature sensitivity in defense responses. In addition, the SNC1 and N proteins had a reduction of nuclear accumulation at elevated temperature, which likely contributes to the inhibition of defense responses. These findings identify a plant temperature sensitive component in disease resistance and provide a potential means to generate plants adapting to a broader temperature range.     

Salicylic Acid and Disease Resistance in Plants

SA has been shown to play an important signaling role in the activation of various plant defense responses following pathogen attack. These responses include the induction of local and systemic disease resistance, the potentiation of host cell death, and the containment of pathogen spread. The mechanisms through which SA mediates these effects are varied and can involve alterations in the activity or synthesis of certain enzymes, increased defense gene expression, potentiation of several defense responses, and/or the generation of free radicals. Through the analysis of mutant plants exhibiting aberrant responses to pathogen infection, many genes encoding products involved in the SA-mediated defense pathway(s) have been isolated. In addition, mounting evidence suggests that certain defense responses can be activated via a SA-independent pathway(s). This review focuses primarily on recent discoveries pertaining to the SA signaling pathway(s) leading to disease resistance; however, a very brief discussion of the SA-independent pathway (s) and its ability to cross-talk with the SA pathway is also presented.     

Yeast Cell Wall Extract Induces Disease Resistance against Bacterial and Fungal Pathogens in Arabidopsis thaliana and Brassica Crop

Housaku Monogatari (HM) is a plant activator prepared from a yeast cell wall extract. We examined the efficacy of HM application and observed that HM treatment increased the resistance of Arabidopsis thaliana and Brassica rapa leaves to bacterial and fungal infections. HM reduced the severity of bacterial leaf spot and anthracnose on A. thaliana and Brassica crop leaves with protective effects. In addition, gene expression analysis of A. thaliana plants after treatment with HM indicated increased expression of several plant defense-related genes. HM treatment appears to induce early activation of jasmonate/ethylene and late activation of salicylic acid (SA) pathways. Analysis using signaling mutants revealed that HM required SA accumulation and SA signaling to facilitate resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola and the fungal pathogen Colletotrichum higginsianum. In addition, HM-induced resistance conferred chitin-independent disease resistance to bacterial pathogens in A. thaliana. These results suggest that HM contains multiple microbe-associated molecular patterns that activate defense responses in plants. These findings suggest that the application of HM is a useful tool that may facilitate new disease control methods.     

Preformed expression of defense is a hallmark of partial resistance to rice blast fungal pathogen <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Background  

Partial resistance to plant pathogens is extensively used in breeding programs since it could contribute to resistance durability. Partial resistance often builds up during plant development and confers quantitative and usually broad-spectrum resistance. However, very little is known on the mechanisms underlying partial resistance. Partial resistance is often explained by poorly effective induction of plant defense systems. By exploring rice natural diversity, we asked whether expression of defense systems before infection could explain partial resistance towards the major fungal pathogen Magnaporthe oryzae. The constitutive expression of 21 defense-related genes belonging to the defense system was monitored in 23 randomly sampled rice cultivars for which partial resistance was measured.