Brendan N. Kidd

MEDIATOR25 Acts as an Integrative Hub for the Regulation of Jasmonate-Responsive Gene Expression in Arabidopsis

The PHYTOCHROME AND FLOWERING TIME1 gene encoding the MEDIATOR25 (MED25) subunit of the eukaryotic Mediator complex is a positive regulator of jasmonate (JA)-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Based on the function of the Mediator complex as a bridge between DNA-bound transcriptional activators and the RNA polymerase II complex, MED25 has been hypothesized to function in association with transcriptional regulators of the JA pathway. However, it is currently not known mechanistically how MED25 functions to regulate JA-responsive gene expression. In this study, we show that MED25 physically interacts with several key transcriptional regulators of the JA signaling pathway, including the APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) transcription factors OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59 and ERF1 as well as the master regulator MYC2. Physical interaction detected between MED25 and four group IX AP2/ERF transcription factors was shown to require the activator interaction domain of MED25 as well as the recently discovered Conserved Motif IX-1/EDLL transcription activation motif of MED25-interacting AP2/ERFs. Using transcriptional activation experiments, we also show that OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59- and ERF1-dependent activation of PLANT DEFENSIN1.2 as well as MYC2-dependent activation of VEGETATIVE STORAGE PROTEIN1 requires a functional MED25. In addition, MED25 is required for MYC2-dependent repression of pathogen defense genes. These results suggest an important role for MED25 as an integrative hub within the Mediator complex during the regulation of JA-associated gene expression.

Auxin Signaling and Transport Promote Susceptibility to the Root-Infecting Fungal Pathogen Fusarium oxysporum in Arabidopsis

Fusarium oxysporum is a root-infecting fungal pathogen that causes wilt disease on a broad range of plant species, including the model plant Arabidopsis thaliana. Currently, very little is known about the molecular or physiological processes that are activated in the host during infection and the roles these processes play in resistance and susceptibility to F. oxysporum. In this study, we analyzed global gene expression profiles of F. oxysporum-infected Arabidopsis plants. Genes involved in jasmonate biosynthesis as well as jasmonate-dependent defense were coordinately induced by F. oxysporum. Similarly, tryptophan pathway genes, including those involved in both indole-glucosinolate and auxin biosynthesis, were upregulated in both the leaves and the roots of inoculated plants. Analysis of plants expressing the DR5:GUS construct suggested that root auxin homeostasis was altered during F. oxysporum infection. However, Arabidopsis mutants with altered auxin and tryptophan-derived metabolites such as indole-glucosinolates and camalexin did not show an altered resistance to this pathogen. In contrast, several auxin-signaling mutants were more resistant to F. oxysporum. Chemical or genetic alteration of polar auxin transport also conferred increased pathogen resistance. Our results suggest that, similarly to many other pathogenic and nonpathogenic or beneficial soil organisms, F. oxysporum requires components of auxin signaling and transport to colonize the plant more effectively. Potential mechanisms of auxin signaling and transport-mediated F. oxysporum susceptibility are discussed.

Linking Jasmonic Acid Signaling, Root Exudates, and Rhizosphere Microbiomes

Jasmonic acid (JA) is an essential hormone in plant development and defense responses in Arabidopsis thaliana. Exogenous treatment with JA has recently been shown to alter root exudate profiles and the composition of root-associated bacterial communities. However, it is currently unknown whether disruptions of the JA in the rhizosphere affect root exudation profiles and the relative abundance of bacteria and archaea in the rhizosphere. In the present study, two Arabidopsis mutants that are disrupted in different branches of the jasmonate pathway, namely myc2 and med25, were cultivated in nutrient solution and soil to profile root exudates and bacterial and archaeal communities, respectively. Compared with the wild type, both mutants showed distinct exudation patterns, including lower amounts of asparagine, ornithine, and tryptophan, as well as distinct bacterial and archaeal community composition, as illustrated by an increased abundance of Streptomyces, Bacillus, and Lysinibacillus taxa in the med25 rhizosphere and an Enterobacteriaceae population in myc2. Alternatively, the Clostridiales population was less abundant in the rhizosphere of both mutants. Similarities between plant genotypes were highly correlated, as determined by operational taxonomic units in the rhizosphere and metabolites in root exudates. This strongly suggests that root exudates play a major role in modulating changes in microbial community composition upon plant defense responses.

Root defense analysis against Fusarium oxysporum reveals new regulators to confer resistance

Fusarium oxysporum is a root-infecting fungal pathogen that causes wilt disease on a broad range of plant species, including Arabidopsis thaliana. Investigation of the defense response against this pathogen had primarily been conducted using leaf tissue and little was known about the root defense response. In this study, we profiled the expression of root genes after infection with F. oxysporum by microarray analysis. In contrast to the leaf response, root tissue did not show a strong induction of defense-associated gene expression and instead showed a greater proportion of repressed genes. Screening insertion mutants from differentially expressed genes in the microarray uncovered a role for the transcription factor ETHYLENE RESPONSE FACTOR72 (ERF72) in susceptibility to F. oxysporum. Due to the role of ERF72 in suppressing programmed cell death and detoxifying reactive oxygen species (ROS), we examined the pub22/pub23/pub24 U-box type E3 ubiquitin ligase triple mutant which is known to possess enhanced ROS production in response to pathogen challenge. We found that the pub22/23/24 mutant is more resistant to F. oxysporum infection, suggesting that a heightened innate immune response provides protection against F. oxysporum. We conclude that root-mediated defenses against soil-borne pathogens can be provided at multiple levels.

Comparative genomics and prediction of conditionally dispensable sequences in legume–infecting Fusarium oxysporum formae speciales facilitates identification of candidate effectors

BackgroundSoil-borne fungi of the Fusarium oxysporum species complex cause devastating wilt disease on many crops including legumes that supply human dietary protein needs across many parts of the globe. We present and compare draft genome assemblies for three legume-infecting formae speciales (ff. spp.): F. oxysporum f. sp. ciceris (Foc-38-1) and f. sp. pisi (Fop-37622), significant pathogens of chickpea and pea respectively, the world’s second and third most important grain legumes, and lastly f. sp. medicaginis (Fom-5190a) for which we developed a model legume pathosystem utilising Medicago truncatula.ResultsFocusing on the identification of pathogenicity gene content, we leveraged the reference genomes of Fusarium pathogens F. oxysporum f. sp. lycopersici (tomato-infecting) and F. solani (pea-infecting) and their well-characterised core and dispensable chromosomes to predict genomic organisation in the newly sequenced legume-infecting isolates. Dispensable chromosomes are not essential for growth and in Fusarium species are known to be enriched in host-specificity and pathogenicity-associated genes. Comparative genomics of the publicly available Fusarium species revealed differential patterns of sequence conservation across F. oxysporum formae speciales, with legume-pathogenic formae speciales not exhibiting greater sequence conservation between them relative to non-legume-infecting formae speciales, possibly indicating the lack of a common ancestral source for legume pathogenicity. Combining predicted dispensable gene content with in planta expression in the model legume-infecting isolate, we identified small conserved regions and candidate effectors, four of which shared greatest similarity to proteins from another legume-infecting ff. spp.ConclusionsWe demonstrate that distinction of core and potential dispensable genomic regions of novel F. oxysporum genomes is an effective tool to facilitate effector discovery and the identification of gene content possibly linked to host specificity. While the legume-infecting isolates didn’t share large genomic regions of pathogenicity-related content, smaller regions and candidate effector proteins were highly conserved, suggesting that they may play specific roles in inducing disease on legume hosts.

Molecular defense responses in roots and the rhizosphere against Fusarium oxysporum

Plants face many different concurrent and consecutive abiotic and biotic stresses during their lifetime. Roots can be infected by numerous pathogens and parasitic organisms. Unlike foliar pathogens, root pathogens have not been explored enough to fully understand root-pathogen interactions and the underlying mechanism of defense and resistance. PR gene expression, structural responses, secondary metabolite and root exudate production, as well as the recruitment of plant defense–assisting “soldier” rhizosphere microbes all assist in root defense against pathogens and herbivores. With new high-throughput molecular tools becoming available and more affordable, now is the opportune time to take a deep look below the ground. In this addendum, we focus on soil-borne Fusarium oxysporum as a pathogen and the options plants have to defend themselves against these hard-to-control pathogens.

Ethylene signaling is important for isoflavonoid-mediated resistance to rhizoctonia solani in roots of medicago truncatula

The root-infecting necrotrophic fungal pathogen Rhizoctoniasolani causes significant disease to all the worlds major food crops. As a model for pathogenesis of legumes, we have examined the interaction of R. solani AG8 with Medicago truncatula. RNAseq analysis of the moderately resistant M. truncatula accession A17 and highly susceptible sickle (skl) mutant (defective in ethylene sensing) identified major early transcriptional reprogramming in A17. Responses specific to A17 included components of ethylene signaling, reactive oxygen species metabolism, and consistent upregulation of the isoflavonoid biosynthesis pathway. Mass spectrometry revealed accumulation of the isoflavonoid-related compounds liquiritigenin, formononetin, medicarpin, and biochanin A in A17. Overexpression of an isoflavone synthase in M. truncatula roots increased isoflavonoid accumulation and resistance to R. solani. Addition of exogenous medicarpin suggested this phytoalexin may be one of several isoflavonoids required to contribute to resistance to R. solani. Together, these results provide evidence for the role of ethylene-mediated accumulation of isoflavonoids during defense against root pathogens in legumes. The involvement of ethylene signaling and isoflavonoids in the regulation of both symbiont-legume and pathogen-legume interactions in the same tissue may suggest tight regulation of these responses are required in the root tissue.

Comparative secretome analysis of Rhizoctonia solani isolates with different host ranges reveals unique secretomes and cell death inducing effectors

Rhizoctonia solani is a fungal pathogen causing substantial damage to many of the worlds’ largest food crops including wheat, rice, maize and soybean. Despite impacting global food security, little is known about the pathogenicity mechanisms employed by R. solani. To enable prediction of effectors possessing either broad efficacy or host specificity, a combined secretome was constructed from a monocot specific isolate, a dicot specific isolate and broad host range isolate infecting both monocot and dicot hosts. Secretome analysis suggested R. solani employs largely different virulence mechanisms to well-studied pathogens, despite in many instances infecting the same host plants. Furthermore, the secretome of the broad host range AG8 isolate may be shaped by maintaining functions for saprophytic life stages while minimising opportunities for host plant recognition. Analysis of possible co-evolution with host plants and in-planta up-regulation in particular, aided identification of effectors including xylanase and inhibitor I9 domain containing proteins able to induce cell death in-planta. The inhibitor I9 domain was more abundant in the secretomes of a wide range of necrotising fungi relative to biotrophs. These findings provide novel targets for further dissection of the virulence mechanisms and potential avenues to control this under-characterised but important pathogen.

Functional metabolomics as a tool to analyze Mediator function and structure in plants

Mediator is a multiprotein transcriptional co-regulator complex composed of four modules; Head, Middle, Tail, and Kinase. It conveys signals from promoter-bound transcriptional regulators to RNA polymerase II and thus plays an essential role in eukaryotic gene regulation. We describe subunit localization and activities of Mediator in Arabidopsis through metabolome and transcriptome analyses from a set of Mediator mutants. Functional metabolomic analysis based on the metabolite profiles of Mediator mutants using multivariate statistical analysis and heat-map visualization shows that different subunit mutants display distinct metabolite profiles, which cluster according to the reported localization of the corresponding subunits in yeast. Based on these results, we suggest localization of previously unassigned plant Mediator subunits to specific modules. We also describe novel roles for individual subunits in development, and demonstrate changes in gene expression patterns and specific metabolite levels in med18 and med25, which can explain their phenotypes. We find that med18 displays levels of phytoalexins normally found in wild type plants only after exposure to pathogens. Our results indicate that different Mediator subunits are involved in specific signaling pathways that control developmental processes and tolerance to pathogen infections.

Belowground Defence Strategies Against Fusarium oxysporum

The root-infecting pathogen Fusarium oxysporum (causative agent of the Fusarium wilt disease) causes widespread losses in many plant species, including important crop plants such as cotton, melons, bananas and tomatoes; many legume species such as chickpeas, peas, lentils and Medicago; and various tree species such as palms. The spores of this pathogen survive in soil for long periods; thus, it is notoriously difficult to eradicate following soil contamination. The pathogen enters into the compatible plants through root tips and lateral root initials, initially invading the cortex tissue. It then gradually moves through the xylem tissue to the upper part of the plant. In addition to the secretion of effectors (e.g. toxins) into the plant cell, the infection by this pathogen can lead to the deposition of plant defence substances such as gums and tyloses in the xylem, which then blocks the water and solute transport to the upper parts of the plant. This leads to wilting, discolouration of xylem, followed by senescence and infection-associated necrotic symptom development in the leaves of infected plants. A number of other developmental changes can also be observed in pathogen-infected plants. Here we describe F. oxysporum–host interactions, highlighting recent updates on pathogen infection strategies and host resistance mechanisms.