Transcriptional reprogramming underpins enhanced plant growth promotion by the biocontrol fungus Trichoderma hamatum GD12 during antagonistic interactions with Sclerotinia sclerotiorum in soil.

The free-living soil fungus Trichoderma hamatum strain GD12 is notable amongst Trichoderma strains in both controlling plant diseases and stimulating plant growth, a property enhanced during its antagonistic interactions with pathogens in soil. These attributes, alongside its markedly expanded genome and proteome compared with other biocontrol and plant growth-promoting Trichoderma strains, imply a rich potential for sustainable alternatives to synthetic pesticides and fertilizers for the control of plant disease and for increasing yields. The purpose of this study was to investigate the transcriptional responses of GD12 underpinning its biocontrol and plant growth promotion capabilities during antagonistic interactions with the pathogen Sclerotinia sclerotiorum in soil. Using an extensive mRNA-seq study capturing different time points during the pathogen-antagonist interaction in soil, we show that dynamic and biphasic signatures in the GD12 transcriptome underpin its biocontrol and plant (lettuce) growth-promoting activities. Functional predictions of differentially expressed genes demonstrate the enrichment of transcripts encoding proteins involved in transportation and oxidation-reduction reactions during both processes and an over-representation of siderophores. We identify a biphasic response during biocontrol characterized by a significant induction of transcripts encoding small-secreted cysteine-rich proteins, secondary metabolite-producing gene clusters and genes unique to GD12. These data support the hypothesis that Sclerotinia biocontrol is mediated by the synthesis and secretion of antifungal compounds and that GD12's unique reservoir of uncharacterized genes is actively recruited during the effective biological control of a plurivorous plant pathogen.

Novel JAZ co-operativity and unexpected JA dynamics underpin Arabidopsis defence responses to Pseudomonas syringae infection.

Pathogens target phytohormone signalling pathways to promote disease. Plants deploy salicylic acid (SA)-mediated defences against biotrophs. Pathogens antagonize SA immunity by activating jasmonate signalling, for example Pseudomonas syringae pv. tomato DC3000 produces coronatine (COR), a jasmonic acid (JA) mimic. This study found unexpected dynamics between SA, JA and COR and co-operation between JAZ jasmonate repressor proteins during DC3000 infection. We used a systems-based approach involving targeted hormone profiling, high-temporal-resolution micro-array analysis, reverse genetics and mRNA-seq. Unexpectedly, foliar JA did not accumulate until late in the infection process and was higher in leaves challenged with COR-deficient P. syringae or in the more resistant JA receptor mutant coi1. JAZ regulation was complex and COR alone was insufficient to sustainably induce JAZs. JAZs contribute to early basal and subsequent secondary plant defence responses. We showed that JAZ5 and JAZ10 specifically co-operate to restrict COR cytotoxicity and pathogen growth through a complex transcriptional reprogramming that does not involve the basic helix-loop-helix transcription factors MYC2 and related MYC3 and MYC4 previously shown to restrict pathogen growth. mRNA-seq predicts compromised SA signalling in a jaz5/10 mutant and rapid suppression of JA-related components on bacterial infection.

Transcriptional Dynamics Driving MAMP-Triggered Immunity and Pathogen Effector-Mediated Immunosuppression in Arabidopsis Leaves Following Infection with Pseudomonas syringae pv tomato DC3000.

Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA- allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae.

Chloroplasts play a central role in plant defence and are targeted by pathogen effectors.

Microbe associated molecular pattern (MAMP) receptors in plants recognize MAMPs and activate basal defences; however a complete understanding of the molecular and physiological mechanisms conferring immunity remains elusive. Pathogens suppress active defence in plants through the combined action of effector proteins. Here we show that the chloroplast is a key component of early immune responses. MAMP perception triggers the rapid, large-scale suppression of nuclear encoded chloroplast-targeted genes (NECGs). Virulent Pseudomonas syringae effectors reprogramme NECG expression in Arabidopsis, target the chloroplast and inhibit photosynthetic CO2 assimilation through disruption of photosystem II. This activity prevents a chloroplastic reactive oxygen burst. These physiological changes precede bacterial multiplication and coincide with pathogen-induced abscisic acid (ABA) accumulation. MAMP pretreatment protects chloroplasts from effector manipulation, whereas application of ABA or the inhibitor of photosynthetic electron transport, DCMU, abolishes the MAMP-induced chloroplastic reactive oxygen burst, and enhances growth of a P. syringae hrpA mutant that fails to secrete effectors.

The salicylic acid dependent and independent effects of NMD in plants.

In eukaryotes, nonsense-mediated mRNA decay (NMD) targets aberrant and selected non-aberrant mRNAs for destruction. A recent screen for mRNAs showing increased abundance in Arabidopsis NMD-deficient mutants revealed that most are associated with the salicylic acid (SA)-mediated defense pathway. mRNAs with conserved peptide upstream open reading frames (CpuORFs or CuORFs) are hugely overrepresented among the smaller class of NMD-regulated transcripts not associated with SA. Here we show that the common phenotypes observed in Arabidopsis NMD mutants are SA-dependent, whereas the upregulation of CpuORF-containing transcripts in NMD mutants is independent of SA. We speculate that CpuORFs could allow the conditional targeting of mRNAs for destruction using the NMD pathway.

A role for nonsense-mediated mRNA decay in plants: pathogen responses are induced in Arabidopsis thaliana NMD mutants.

Nonsense-mediated mRNA decay (NMD) is a conserved mechanism that targets aberrant mRNAs for destruction. NMD has also been found to regulate the expression of large numbers of genes in diverse organisms, although the biological role for this is unclear and few evolutionarily conserved targets have been identified. Expression analyses of three Arabidopsis thaliana lines deficient in NMD reveal that the vast majority of NMD-targeted transcripts are associated with response to pathogens. Congruently, NMD mutants, in which these transcripts are elevated, confer partial resistance to Pseudomonas syringae. These findings suggest a biological rationale for the regulation of gene expression by NMD in plants and suggest that manipulation of NMD could offer a new approach for crop protection. Amongst the few non-pathogen responsive NMD-targeted genes, one potential NMD targeted signal, the evolutionarily conserved upstream open reading frame (CuORF), was found to be hugely over-represented, raising the possibility that this feature could be used to target specific physiological mRNAs for control by NMD.

Antagonism between salicylic and abscisic acid reflects early host-pathogen conflict and moulds plant defence responses.

The importance of phytohormone balance is increasingly recognized as central to the outcome of plant-pathogen interactions. Recently it has been demonstrated that abscisic acid signalling pathways are utilized by the bacterial phytopathogen Pseudomonas syringae to promote pathogenesis. In this study, we examined the dynamics, inter-relationship and impact of three key acidic phytohormones, salicylic acid, abscisic acid and jasmonic acid, and the bacterial virulence factor, coronatine, during progression of P. syringae infection of Arabidopsis thaliana. We show that levels of SA and ABA, but not JA, appear to play important early roles in determining the outcome of the infection process. SA is required in order to mount a full innate immune responses, while bacterial effectors act rapidly to activate ABA biosynthesis. ABA suppresses inducible innate immune responses by down-regulating SA biosynthesis and SA-mediated defences. Mutant analyses indicated that endogenous ABA levels represent an important reservoir that is necessary for effector suppression of plant-inducible innate defence responses and SA synthesis prior to subsequent pathogen-induced increases in ABA. Enhanced susceptibility due to loss of SA-mediated basal resistance is epistatically dominant over acquired resistance due to ABA deficiency, although ABA also contributes to symptom development. We conclude that pathogen-modulated ABA signalling rapidly antagonizes SA-mediated defences. We predict that hormonal perturbations, either induced or as a result of environmental stress, have a marked impact on pathological outcomes, and we provide a mechanistic basis for understanding priming events in plant defence.

Transcriptional regulation by an NAC (NAM-ATAF1,2-CUC2) transcription factor attenuates ABA signalling for efficient basal defence towards Blumeria graminis f. sp. hordei in Arabidopsis.

ATAF1 is a member of a largely uncharacterized plant-specific gene family encoding NAC transcription factors, and is induced in response to various abiotic and biotic stimuli in Arabidopsis thaliana. Previously, we showed that a mutant allele of ATAF1 compromises penetration resistance in Arabidopsis with respect to the non-host biotrophic pathogen Blumeria graminis f. sp. hordei (Bgh). In this study, we have used genome-wide transcript profiling to characterize signalling perturbations in ataf1 plants following Bgh inoculation. Comparative transcriptomic analyses identified an over-representation of abscisic acid (ABA)-responsive genes, including the ABA biosynthesis gene AAO3, which is significantly induced in ataf1 plants compared to wild-type plants following inoculation with Bgh. Additionally, we show that Bgh inoculation results in decreased endogenous ABA levels in an ATAF1-dependent manner, and that the ABA biosynthetic mutant aao3 showed increased penetration resistance to Bgh compared to wild-type plants. Furthermore, we show that ataf1 plants show ABA-hyposensitive phenotypes during seedling development and germination. Our data support a negative correlation between ABA levels and penetration resistance, and identify ATAF1 as a new stimuli-dependent attenuator of ABA signalling for the mediation of efficient penetration resistance in Arabidopsis upon Bgh attack.

Pseudomonas syringae pv. tomato hijacks the Arabidopsis abscisic acid signalling pathway to cause disease.

We have found that a major target for effectors secreted by Pseudomonas syringae is the abscisic acid (ABA) signalling pathway. Microarray data identified a prominent group of effector-induced genes that were associated with ABA biosynthesis and also responses to this plant hormone. Genes upregulated by effector delivery share a 42% overlap with ABA-responsive genes and are also components of networks induced by osmotic stress and drought. Strongly induced were NCED3, encoding a key enzyme of ABA biosynthesis, and the abscisic acid insensitive 1 (ABI1) clade of genes encoding protein phosphatases type 2C (PP2Cs) involved in the regulation of ABA signalling. Modification of PP2C expression resulting in ABA insensitivity or hypersensitivity led to restriction or enhanced multiplication of bacteria, respectively. Levels of ABA increased rapidly during bacterial colonisation. Exogenous ABA application enhanced susceptibility, whereas colonisation was reduced in an ABA biosynthetic mutant. Expression of the bacterial effector AvrPtoB in planta modified host ABA signalling. Our data suggest that a major virulence strategy is effector-mediated manipulation of plant hormone homeostasis, which leads to the suppression of defence responses.

RIN13 is a positive regulator of the plant disease resistance protein RPM1.

The RPM1 protein confers resistance to Pseudomonas syringae pv tomato DC3000 expressing either of the Type III effector proteins AvrRpm1 or AvrB. Here, we describe the isolation and functional characterization of RPM1 Interacting Protein 13 (RIN13), a resistance protein interactor shown to positively enhance resistance function. Ectopic expression of RIN13 (RIN13s) enhanced bacterial restriction mechanisms but paradoxically abolished the normally rapid hypersensitive response (HR) controlled by RPM1. In contrast with wild-type plants, leaves expressing RIN13s did not undergo electrolyte leakage or accumulate H2O2 after bacterial delivery of AvrRpm1. Overexpression of RIN13 also altered the transcription profile observed during a normal HR. By contrast, RIN13 knockout plants had the same ion leakage signatures and HR timing of wild-type plants in response to DC3000(avrRpm1) but failed to suppress bacterial growth. The modified phenotypes seen in the RIN13s/as plants were specific to recognition of AvrRpm1 or AvrB, and wild-type responses were observed after challenge with other incompatible pathogens or the virulent DC3000 isolate. Our results suggest that cell death is not necessary to confer resistance, and engineering enhanced resistance without activation of programmed cell death is a real possibility.

Storekeeper defines a new class of plant-specific DNA-binding proteins and is a putative regulator of patatin expression.

The expression of class I patatin genes is restricted to potato tubers but can be induced in other tissues by exogenous sucrose. Here we show that tuber-specific and sucrose-inducible gene expression is reduced in transgenic potato plants by mutations in a conserved 10 base pair motif within the B-box of the patatin promoter. In a southwestern screen, we have isolated a novel DNA-binding protein designated Storekeeper (STK) that specifically recognises the B-box motif in vitro. Gel shift experiments with an STK-specific antibody suggest that STK is the B-box binding protein found in tuber nuclei. We propose that STK, the defining member of a new class of DNA binding proteins, regulates patatin expression in potato tubers via the B-box motif.