The ethylene response factor (ERF) family in Arabidopsis thaliana comprises 122 members in 12 groups, yet the biological functions of the majority remain unknown. Of the group IX ERFs, the IXc subgroup has been studied the most, and includes ERF1, ERF14 and ORA59, which play roles in plant innate immunity. Here we investigate the biological functions of two members of the less studied IXb subgroup: ERF5 and ERF6. In order to identify potential targets of these transcription factors, microarray analyses were performed on plants constitutively expressing either ERF5 or ERF6. Expression of defense genes, JA/Et-responsive genes and genes containing the GCC box promoter motif were significantly upregulated in both ERF5 and ERF6 transgenic plants, suggesting that ERF5 and ERF6 may act as positive regulators of JA-mediated defense and potentially overlap in their function. Since defense against necrotrophic pathogens is generally mediated through JA/Et-signalling, resistance against the fungal necrotroph Botrytis cinerea was examined. Constitutive expression of ERF5 or ERF6 resulted in significantly increased resistance. Although no significant difference in susceptibility to B. cinerea was observed in either erf5 or erf6 mutants, the erf5 erf6 double mutant showed a significant increase in susceptibility, which was likely due to compromised JA-mediated gene expression, since JA-induced gene expression was reduced in the double mutant. Taken together these data suggest that ERF5 and ERF6 play positive but redundant roles in defense against B. cinerea. Since mutual antagonism between JA/Et and salicylic acid (SA) signalling is well known, the UV-C inducibility of an SA-inducible gene, PR-1, was examined. Reduced inducibilty in both ERF5 and ERF6 constitutive overexepressors was consistent with suppression of SA-mediated signalling, as was an increased susceptibility to avirulent Pseudomonas syringae. These data suggest that ERF5 and ERF6 may also play a role in the antagonistic crosstalk between the JA/Et and SA signalling pathways.

The importance of light with respect to the outcome of plant-pathogen interactions is becoming increasingly evident: light affects both the host response and the virulence of some pathogens. The response of plants to environmental signals and stresses is modulated by the circadian clock, and it is apparent that this may include immune responses. Photo and temporal regulation of immune responses may allow plants to anticipate and react more effectively to particular pathogen infections. These aspects of regulation are sometimes overlooked when designing experiments to understand plantpathogen interactions, complicating the interpretation of the outcomes and the direct comparisons of studies. We review recent key findings in these areas and discuss the implications for experimental design and analyses.

The circadian clock allows plants to anticipate predictable daily changes in abiotic stimuli, such as light; however, whether the clock similarly allows plants to anticipate interactions with other organisms is unknown. Here we show that Arabidopsis thaliana (Arabidopsis) has circadian clock-mediated variation in resistance to the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), with plants being least susceptible to infection in the subjective morning. We suggest that the increased resistance to Pst DC3000 observed in the morning in Col-0 plants results from clock-mediated modulation of pathogen associated molecular pattern (PAMP)-triggered immunity. Analysis of publicly available microarray data revealed that a large number of Arabidopsis defence-related genes showed both diurnal- and circadian-regulation, including genes involved in the perception of the PAMP flagellin which exhibit a peak in expression in the morning. Accordingly, we observed that PAMP-triggered callose deposition was significantly higher in wild-type plants inoculated with Pst DC3000 hrpA in the subjective morning than in the evening, while no such temporal difference was evident in arrhythmic plants. Our results suggest that PAMP-triggered immune responses are modulated by the circadian clock and that temporal regulation allows plants to anticipate and respond more effectively to pathogen challenges in the daytime.

The desiccation-tolerant phenotype of angiosperm resurrection plants is thought to rely on the induction of protective mechanisms that maintain cellular integrity during water loss. Two-dimensional (2D) sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the Xerophyta viscosa Baker proteome was carried out during dehydration to identify proteins that may play a role in such mechanisms. Quantitative analysis revealed a greater number of changes in protein expression levels at 35% than at 65% relative water content (RWC) compared to fully hydrated plants, and 17 dehydrationresponsive proteins were identified by tandem mass spectrometry (MS). Proteins showing increased abundance during drying included an RNA-binding protein, chloroplast FtsH protease, glycolytic enzymes and antioxidants. A number of photosynthetic proteins declined sharply in abundance in X. viscosa at RWC below 65%, including four components of photosystem II (PSII), and Western blot analysis confirmed that two of these (psbP and Lhcb2) were not detectable at 30% RWC. These data confirm that poikilochlorophylly in X. viscosa involves the breakdown of photosynthetic proteins during dismantling of the thylakoid membranes. In contrast, levels of these photosynthetic proteins were largely maintained during dehydration in the homoiochlorophyllous species Craterostigma plantagineum Hochst, which does not dismantle thylakoid membranes on drying.

Basal resistance is the ultimately unsuccessful plant defense response to infection with a virulent pathogen. It is thought to be triggered by host recognition of pathogen-associated molecular patterns, with subsequent suppression of particular components by pathogen effectors. To identify novel components of Arabidopsis basal resistance against the bacterial pathogen Pseudomonas syringae pv. tomato, microarray expression profiling was carried out on the cirl mutant, which displays enhanced resistance against P. syringae pv. tomato. This identified two genes, At4g23810 and At2g40000, encoding the transcription factor WRKY53 and the nematode resistance protein-like HSPRO2, whose expression was upregulated in cir1 prior to pathogen infection and in wild-type plants after P. syringae pv. tomato infection. WRKY53 and HSPRO2 are positive regulators of basal resistance. Knockout mutants of both genes were more susceptible to P. syringae pv. tomato infection than complemented lines, with increased growth of the pathogen in planta. WRKY53 and HSPRO2 appear to function downstream of salicylic acid and to be negatively regulated by signaling through jasmonic acid and ethylene.

Plants that hyperaccumulate Ni exhibit an exceptional degree of Ni tolerance and the ability to translocate Ni in large amounts from root to shoot. In hyperaccumulator plants in the genus Alyssum, free His is an important Ni binding ligand that increases in the xylem proportionately to root Ni uptake. To determine the molecular basis of the His response and its contribution to Ni tolerance, transcripts representing seven of the eight enzymes involved in His biosynthesis were investigated in the hyperaccumulator species Alyssum lesbiacum by RNA gel blot analysis. None of the transcripts changed in abundance in either root or shoot tissue when plants were exposed to Ni, but transcript levels were constitutively higher in A. lesbiacum than in the congeneric nonaccumulator A. montanum, especially for the first enzyme in the biosynthetic pathway, ATP-phosphoribosyltransferase (ATP-PRT). Comparison with the weak hyperaccumulator A. serpyllifolium revealed a close correlation between Ni tolerance, root His concentration, and ATP-PRT transcript abundance. Overexpression of an A. lesbiacum ATP-PRT cDNA in transgenic Arabidopsis thaliana increased the pool of free His up to 15-fold in shoot tissue, without affecting the concentration of any other amino acid. His-overproducing lines also displayed elevated tolerance to Ni but did not exhibit increased Ni concentrations in either xylem sap or shoot tissue, suggesting that additional factors are necessary to recapitulate the complete hyperaccumulator phenotype. These results suggest that ATP-PRT expression plays a major role in regulating the pool of free His and contributes to the exceptional Ni tolerance of hyperaccumulator Alyssum species.

Plants have evolved systems analogous to animal innate immunity that recognise pathogen-associated molecular patterns (PAMPs). PAMP detection is an important component of non-host resistance in plants and serves as an early warning system for the presence of potential pathogens. Binding of a PAMP to the appropriate pattern recognition receptor leads to downstream signalling events and, ultimately, to the induction of basal defence systems. To overcome non-host resistance, pathogens have evolved effectors that target specific regulatory components of the basal defence system. In turn, this has led to the evolution in plants of cultivar-specific resistance mediated by R proteins, which guard the targets of effectors against pathogen manipulation; the arms race continues.

SummaryThe circadian clock, an internal time‐keeping mechanism, allows plants to anticipate regular changes in the environment, such as light and dark, and biotic challenges such as pathogens and herbivores. Here, we demonstrate that the plant circadian clock influences susceptibility to the necrotrophic fungal pathogen, Botrytis cinerea. Arabidopsis plants show differential susceptibility to B. cinerea depending on the time of day of inoculation. Decreased susceptibility after inoculation at dawn compared with night persists under constant light conditions and is disrupted in dysfunctional clock mutants, demonstrating the role of the plant clock in driving time‐of‐day susceptibility to B. cinerea. The decreased susceptibility to B. cinerea following inoculation at subjective dawn was associated with faster transcriptional reprogramming of the defence response with gating of infection‐responsive genes apparent. Direct target genes of core clock regulators were enriched among the transcription factors that responded more rapidly to infection at subjective dawn than subjective night, suggesting an influence of the clock on the defence‐signalling network. In addition, jasmonate signalling plays a crucial role in the rhythmic susceptibility of Arabidopsis to B. cinerea with the enhanced susceptibility to this pathogen at subjective night lost in a jaz6 mutant.