Dual regulation of gene expression mediated by extended MAPK activation and salicylic acid contributes to robust innate immunity in Arabidopsis thaliana.

Network robustness is a crucial property of the plant immune signaling network because pathogens are under a strong selection pressure to perturb plant network components to dampen plant immune responses. Nevertheless, modulation of network robustness is an area of network biology that has rarely been explored. While two modes of plant immunity, Effector-Triggered Immunity (ETI) and Pattern-Triggered Immunity (PTI), extensively share signaling machinery, the network output is much more robust against perturbations during ETI than PTI, suggesting modulation of network robustness. Here, we report a molecular mechanism underlying the modulation of the network robustness in Arabidopsis thaliana. The salicylic acid (SA) signaling sector regulates a major portion of the plant immune response and is important in immunity against biotrophic and hemibiotrophic pathogens. In Arabidopsis, SA signaling was required for the proper regulation of the vast majority of SA-responsive genes during PTI. However, during ETI, regulation of most SA-responsive genes, including the canonical SA marker gene PR1, could be controlled by SA-independent mechanisms as well as by SA. The activation of the two immune-related MAPKs, MPK3 and MPK6, persisted for several hours during ETI but less than one hour during PTI. Sustained MAPK activation was sufficient to confer SA-independent regulation of most SA-responsive genes. Furthermore, the MPK3 and SA signaling sectors were compensatory to each other for inhibition of bacterial growth as well as for PR1 expression during ETI. These results indicate that the duration of the MAPK activation is a critical determinant for modulation of robustness of the immune signaling network. Our findings with the plant immune signaling network imply that the robustness level of a biological network can be modulated by the activities of network components.

Spatio-temporal expression patterns of Arabidopsis thaliana and Medicago truncatula defensin-like genes.

Plant genomes contain several hundred defensin-like (DEFL) genes that encode short cysteine-rich proteins resembling defensins, which are well known antimicrobial polypeptides. Little is known about the expression patterns or functions of many DEFLs because most were discovered recently and hence are not well represented on standard microarrays. We designed a custom Affymetrix chip consisting of probe sets for 317 and 684 DEFLs from Arabidopsis thaliana and Medicago truncatula, respectively for cataloging DEFL expression in a variety of plant organs at different developmental stages and during symbiotic and pathogenic associations. The microarray analysis provided evidence for the transcription of 71% and 90% of the DEFLs identified in Arabidopsis and Medicago, respectively, including many of the recently annotated DEFL genes that previously lacked expression information. Both model plants contain a subset of DEFLs specifically expressed in seeds or fruits. A few DEFLs, including some plant defensins, were significantly up-regulated in Arabidopsis leaves inoculated with Alternaria brassicicola or Pseudomonas syringae pathogens. Among these, some were dependent on jasmonic acid signaling or were associated with specific types of immune responses. There were notable differences in DEFL gene expression patterns between Arabidopsis and Medicago, as the majority of Arabidopsis DEFLs were expressed in inflorescences, while only a few exhibited root-enhanced expression. By contrast, Medicago DEFLs were most prominently expressed in nitrogen-fixing root nodules. Thus, our data document salient differences in DEFL temporal and spatial expression between Arabidopsis and Medicago, suggesting distinct signaling routes and distinct roles for these proteins in the two plant species.

Metabolite profiling of Arabidopsis inoculated with Alternaria brassicicola reveals that ascorbate reduces disease severity.

The interaction between the pathogenic ascomycete Alternaria brassicicola and Arabidopsis was investigated by metabolite profiling. The effect of A. brassicicola challenge on metabolite levels was substantial, with nearly 50% of detected compounds undergoing significant changes. Mutations blocking ethylene, jasmonic acid, or ethylene signaling had little effect on metabolite levels. The effects of altering levels of some metabolites were tested by exogenous application during A. brassicicola inoculation. Gamma amino-butyric acid (GABA) or xylitol promoted, while trehalose and ascorbate inhibited, disease severity. GABA promoted, and ascorbate strongly inhibited, fungal growth in culture. Arabidopsis vtc1 and vtc2 mutants, that have low levels of ascorbate, were more susceptible to A. brassicicola. Ascorbate levels declined following A. brassicicola inoculation while levels of dehydroascorbate increased, resulting in a shift of the redox balance between these compounds in the direction of oxidation. These results demonstrate that ascorbate is an important component of resistance to this pathogen.

Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus.

Plant and animal perception of microbes through pathogen surveillance proteins leads to MAP kinase signalling and the expression of defence genes. However, little is known about how plant MAP kinases regulate specific gene expression. We report that, in the absence of pathogens, Arabidopsis MAP kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further supported by the suppression of PAD3 expression in mpk4-wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation.

Arabidopsis cytochrome P450 monooxygenase 71A13 catalyzes the conversion of indole-3-acetaldoxime in camalexin synthesis.

Camalexin (3-thiazol-2-yl-indole) is an indole alkaloid phytoalexin produced by Arabidopsis thaliana that is thought to be important for resistance to necrotrophic fungal pathogens, such as Alternaria brassicicola and Botrytis cinerea. It is produced from Trp, which is converted to indole acetaldoxime (IAOx) by the action of cytochrome P450 monooxygenases CYP79B2 and CYP79B3. The remaining biosynthetic steps are unknown except for the last step, which is conversion of dihydrocamalexic acid to camalexin by CYP71B15 (PAD3). This article reports characterization of CYP71A13. Plants carrying cyp71A13 mutations produce greatly reduced amounts of camalexin after infection by Pseudomonas syringae or A. brassicicola and are susceptible to A. brassicicola, as are pad3 and cyp79B2 cyp79B3 mutants. Expression levels of CYP71A13 and PAD3 are coregulated. CYP71A13 expressed in Escherichia coli converted IAOx to indole-3-acetonitrile (IAN). Expression of CYP79B2 and CYP71A13 in Nicotiana benthamiana resulted in conversion of Trp to IAN. Exogenously supplied IAN restored camalexin production in cyp71A13 mutant plants. Together, these results lead to the conclusion that CYP71A13 catalyzes the conversion of IAOx to IAN in camalexin synthesis and provide further support for the role of camalexin in resistance to A. brassicicola.

Phenetic relationships among different races of Striga gesnerioides (Willd.) Vatke from West Africa.

Striga gesnerioides is a root hemiparasite that primarily parasitizes dicotyledonous species, including cowpea (Vigna unguiculata L.) and other legumes. Based on the differential resistance response of various cultivars, landraces, and breeding lines, it has been proposed that several distinct races of cowpea-parasitic S. gesnerioides exist in West Africa. In this study, we used amplified fragment length polymorphism profile analysis to examine the genetic variability within and among populations of cowpea-parasitic S. gesnerioides within the suspected distribution range of a particular race, and statistical clustering methods to define the phenetic relationships of the various races in West Africa. Our data indicate that genetic variability within and among populations of each of the previously recognized races of cowpea-parasitic S. gesnerioides is extremely low. On the basis of genotypic profile and host differential resistance responses, 2 previously unknown races were identified. Of the 7 races now identifiable, races SG1 (from Burkina Faso) and SG5 (from Cameroon) are the most closely related, and SG4 (from Benin) and SG3 (from Niger/Nigeria) are the most divergent. SG6, a new race of the parasite identified in Senegal, was found to be the most genetically similar to SG4 from Benin. We also demonstrate that a hypervirulent isolate of the S. gesnerioides from Zakpota, in the Republic of Benin, is genotypically distinct from other populations of SG4, thereby warranting designation as a separate race, which we called SG4z. To further support our race classification scheme, we identified a group of molecular markers that effectively discriminate each of the various races. Finally, we show that an isolate (designated SG4i) of the wild legume Indigofera hirsuta--parasitic S. gesnerioides is genetically distinct and significantly diverged from the various races of cowpea-parasitic S. gesnerioides. Our data suggest that both geographic isolation and host-driven selection are critical factors defining race formation in S. gesnerioides in West Africa.

Genetic Structure and Analysis of Host and Nonhost Interactions of Striga gesnerioides (Witchweed) from Central Florida.

ABSTRACT Striga gesnerioides is a root hemiparasite of wild and cultivated legumes, among which cowpea (Vigna unguiculata) and Indigofera hirsuta are suitable hosts. In this study, we examined the genetic structure and host-parasite interaction of a strain of S. gesnerioides parasitizing I. hirsuta (SGFL) from central Florida (United States). Amplified fragment length polymorphism analysis was used to compare genetic profiles from 71 individual S. gesnerioides plants (SGFL) representing four different populations in central Florida. Our results showed that these individuals are genetically similar, with pairwise genetic distances ranging from 0.00 to 0.037. A cluster analysis grouped all four S. gesnerioides populations from Florida, separating them from S. gesnerioides isolates parasitic on I. hirsuta and cowpea collected from West Africa. One EcoRI and MseI selective primer combination generated a 510-bp fragment present in individuals from the SGFL and the West African isolate parasitic on I. hirsuta, but absent in isolates parasitic on cowpea. Germination of seed from individuals of all four populations of S. gesnerioides parasitic on I. hirsuta from Florida was stimulated by root exudates from cowpea genotypes Blackeye and TVX-3236, known to be highly susceptible to all races of S. gesnerioides parasitic on cowpea in West Africa. SGFL seedlings failed to parasitize cowpea, with the development of attached SGFL seedlings arrested at the tubercle stage. The very high level of genetic uniformity observed within and among the central Florida populations suggests that there was likely a single introduction of the parasite or strong host-driven selection to genetic uniformity.These findings are important in assessing the potential of the parasite as an agronomically significant pest in the United States.