Mark H. Bennett

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.

Arabidopsis systemic immunity uses conserved defense signaling pathways and is mediated by jasmonates

In the absence of adaptive immunity displayed by animals, plants respond locally to biotic challenge via inducible basal defense networks activated through recognition and response to conserved pathogen-associated molecular patterns. In addition, immunity can be induced in tissues remote from infection sites by systemic acquired resistance (SAR), initiated after gene-for-gene recognition between plant resistance proteins and microbial effectors. The nature of the mobile signal and remotely activated networks responsible for establishing SAR remain unclear. Salicylic acid (SA) participates in the local and systemic response, but SAR does not require long-distance translocation of SA. Here, we show that, despite the absence of pathogen-associated molecular pattern contact, systemically responding leaves rapidly activate a SAR transcriptional signature with strong similarity to local basal defense. We present several lines of evidence that suggest jasmonates are central to systemic defense, possibly acting as the initiating signal for classic SAR. Jasmonic acid (JA), but not SA, rapidly accumulates in phloem exudates of leaves challenged with an avirulent strain of Pseudomonas syringae. In systemically responding leaves, transcripts associated with jasmonate biosynthesis are up-regulated within 4 h, and JA increases transiently. SAR can be mimicked by foliar JA application and is abrogated in mutants impaired in jasmonate synthesis or response. We conclude that jasmonate signaling appears to mediate long-distance information transmission. Moreover, the systemic transcriptional response shares extraordinary overlap with local herbivory and wounding responses, indicating that jasmonates may be pivotal to an evolutionarily conserved signaling network that decodes multiple abiotic and biotic stress signals.

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.

Cell Wall Damage-Induced Lignin Biosynthesis Is Regulated by a Reactive Oxygen Species- and Jasmonic Acid-Dependent Process in Arabidopsis

The plant cell wall is a dynamic and complex structure whose functional integrity is constantly being monitored and maintained during development and interactions with the environment. In response to cell wall damage (CWD), putatively compensatory responses, such as lignin production, are initiated. In this context, lignin deposition could reinforce the cell wall to maintain functional integrity. Lignin is important for the plant’s response to environmental stress, for reinforcement during secondary cell wall formation, and for long-distance water transport. Here, we identify two stages and several components of a genetic network that regulate CWD-induced lignin production in Arabidopsis (Arabidopsis thaliana). During the early stage, calcium and diphenyleneiodonium-sensitive reactive oxygen species (ROS) production are required to induce a secondary ROS burst and jasmonic acid (JA) accumulation. During the second stage, ROS derived from the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D and JA-isoleucine generated by JASMONIC ACID RESISTANT1, form a negative feedback loop that can repress each other’s production. This feedback loop in turn seems to influence lignin accumulation. Our results characterize a genetic network enabling plants to regulate lignin biosynthesis in response to CWD through dynamic interactions between JA and ROS.

Modifications to the Arabidopsis Defense Proteome Occur Prior to Significant Transcriptional Change in Response to Inoculation with Pseudomonas syringae

Alterations in the proteome of Arabidopsis (Arabidopsis thaliana) leaves during responses to challenge by Pseudomonas syringae pv tomato DC3000 were analyzed using two-dimensional gel electrophoresis. Protein changes characteristic of the establishment of disease, basal resistance, and resistance-gene-mediated resistance were examined by comparing responses to DC3000, a hrp mutant, and DC3000 expressing avrRpm1, respectively. The abundance of each protein identified was compared with that of selected transcripts obtained from comparable GeneChip experiments. We report changes in three subcellular fractions: total soluble protein, chloroplast enriched, and mitochondria enriched over four time points (1.5–6 h after inoculation). In total, 73 differential spots representing 52 unique proteins were successfully identified. Many of the changes in protein spot density occurred before significant transcriptional reprogramming was evident between treatments. The high proportion of proteins represented by more than one spot indicated that many of the changes to the proteome can be attributed to posttranscriptional modifications. Proteins found to show significant change after bacterial challenge are representative of two main functional groups: defense-related antioxidants and metabolic enzymes. Significant changes to photosystem II and to components of the mitochondrial permeability transition were also identified. Rapid communication between organelles and regulation of primary metabolism through redox-mediated signaling are supported by our data.

Hrp mutant of Pseudomonas syringae pv Phaseolicola induces cell wall alterations but not membrane damage leading to the hypersensitive reaction in lettuce

Both wild-type (S21-WT) and hrpD- (S21–533) strains of Pseudomonas syringae pv phaseolicola induced the formation of large paramural papillae in lettuce (Lactuca sativa) mesophyll cells adjacent to bacterial colonies. Localized alterations to the plant cell wall included deposition of hydroxyproline-rich glycoproteins, phe-nolics, and callose, and were associated with proliferation of the endoplasmic reticulum and multivesicular bodies. Tissue collapse during the hypersensitive reaction caused by S21-WT was associated with electrolyte leakage and rapid accumulation of the phy-toalexin lettucenin A, both of which followed membrane damage indicated by the failure of mesophyll cells to plasmolyze. A few cells lost the ability to plasmolyze after inoculation with S21–533, and low levels of lettucenin A were recorded, but neither leakage of electrolytes nor tissue collapse were detected. Dysfunction of the plasma membrane in cells adjacent to colonies of S21-WT led to extensive vacuolation of the cytoplasm, organelle disruption, and cytoplasmic collapse[mdash]changes unlike those occurring in cells undergoing apoptosis. Strain S21–533 remained viable within symptomless tissue, whereas cells of S21-WT were killed as a consequence of the hypersensitive reaction. Our observations emphasize the subtle coordination of the plants response occurring at the subcellular level.

A rapid and robust method for simultaneously measuring changes in the phytohormones ABA, JA and SA in plants following biotic and abiotic stress.

We describe an efficient method for the rapid quantitative determination of the abundance of three acidic plant hormones from a single crude extract directly by LC/MS/MS. The method exploits the sensitivity of MS and uses multiple reaction monitoring and isotopically labelled samples to quantify the phytohormones abscisic acid, jasmonic acid and salicylic acid in Arabidopsis leaf tissue.

The metabolic transition during disease following infection of Arabidopsis thaliana by Pseudomonas syringae pv. tomato.

The outcome of bacterial infection in plants is determined by the ability of the pathogen to successfully occupy the apoplastic space and deliver a constellation of effectors that collectively suppress basal and effector-triggered immune responses. In this study, we examined the metabolic changes associated with establishment of disease using analytical techniques that interrogated a range of chemistries. We demonstrated clear differences in the metabolome of Arabidopsis thaliana leaves infected with virulent Pseudomonas syringae within 8 h of infection. In addition to confirmation of changes in phenolic and indolic compounds, we identified rapid alterations in the abundance of amino acids and other nitrogenous compounds, specific classes of glucosinolates, disaccharides, and molecules that influence the prevalence of reactive oxygen species. Our data illustrate that, superimposed on defence suppression, pathogens reconfigure host metabolism to provide the sustenance required to support exponentially growing populations of apoplastically localized bacteria. We performed a detailed baseline study reporting the metabolic dynamics associated with bacterial infection. Moreover, we have integrated these data with the results of transcriptome profiling to distinguish metabolomic pathways that are transcriptionally activated from those that are post-transcriptionally regulated.

Disruption of Two Defensive Signaling Pathways by a Viral RNA Silencing Suppressor

The Cucumber mosaic virus (CMV) 2b counter-defense protein disrupts plant antiviral mechanisms mediated by RNA silencing and salicylic acid (SA). We used microarrays to investigate defensive gene expression in 2b-transgenic Arabidopsis thaliana plants. Surprisingly, 2b inhibited expression of few SA-regulated genes and, in some instances, enhanced the effect of SA on certain genes. Strikingly, the 2b protein inhibited changes in the expression of 90% of genes regulated by jasmonic acid (JA). Consistent with this, infection of plants with CMV, but not the 2b gene-deletion mutant CMVDelta2b, strongly inhibited JA-inducible gene expression. JA levels were unaffected by infection with either CMV or CMVDelta2b. Although the CMV-Arabidopsis interaction is a compatible one, SA accumulation, usually considered to be an indicator of plant resistance, was increased in CMV-infected plants but not in CMVDelta2b-infected plants. Thus, the 2b protein inhibits JA signaling at a step downstream of JA biosynthesis but it primes induction of SA biosynthesis by another CMV gene product or by the process of infection itself. Like many plant viruses, CMV is aphid transmitted. JA is important in plant defense against insects. This raises the possibility that disruption of JA-mediated gene expression by the 2b protein may influence CMV transmission by aphids.

Identification of cell-wall stress as a hexose-dependent and osmosensitive regulator of plant responses.

Development, abiotic and biotic stress each affect the physical architecture and chemical composition of the plant cell wall, making maintenance of cell-wall integrity an important component of many plant processes. Cellulose biosynthesis inhibition (CBI) was employed to impair the functional integrity of the cell wall, and the plants response to this specific stress was characterized in an Arabidopsis seedling model system. CBI caused changes in the expression of genes involved in mechanoperception, the response to microbial challenge, and lignin and cell-wall polysaccharide biosynthesis. Following CBI, activation of a UDP-D-xylose 4-epimerase gene correlated with increases in arabinose and uronic acid content in seedling cell walls. Activation of pathogen response genes, lignin deposition and lesion formation were dependent on externally supplied sugars and were suppressed by osmotic support. Lignin deposition in the root elongation zone caused by CBI was reduced in atrbohd (NADPH oxidase) mutant seedlings but increased in jasmonic acid resistant1 (jar1-1) mutant seedlings. Phytohormone measurements showed that CBI-induced increases in jasmonic (JA) and salicylic acids were dependent on sugar availability and prevented by osmotic support. We show that CBI activates responses commonly attributed to both abiotic and microbial challenges. Glucose/sucrose and turgor pressure are critical components in maintenance of cell-wall integrity and the regulation of induced responses, including JA biosynthesis. Lignin deposition induced by CBI is regulated by JAR1-1 and NADPH oxidase-dependent signalling processes. Our results identify components of the mechanism that mediates the response to impairment of cell-wall integrity in Arabidopsis thaliana.