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Expression of the French bean chalcone synthase ( CHS ) gene is induced in response to external stimuli and to the plant's tissue-specific and developmental program. We have previously demonstrated the essential role of a cooperative regulatory element (CAR), containing a G-box and H-box, in tissue-specific expression. In this paper, we focus on understanding the requirement for cis -elements in the response of the CHS15 promoter to the abiotic elicitor HgCl 2 , mechanical wounding and infection with tobacco mosaic virus (TMV). Leaves of tobacco plants transformed with a chimeric CHS15 promoter-β-glucuronidase gene construct carrying a mutation at the G-box did not exhibit impaired promoter response to wounding, but did show a 19% reduction in the response to HgCl 2 and TMV. A mutation at the H-box within CAR resulted in a 30% increase in promoter response to wounding and reductions of 36 and 54% in the response to HgCl 2 and TMV, respectively. These results suggest differential utilization of regulatory cis -elements for stress-induced and tissue-specific expression of the bean CHS15 promoter.

The G-box (CACGTG) and H-box (CCTACC) cis elements function in the activation of phenylpropanoid biosynthetic genes involved in the elaboration of lignin precursors, phytoalexins and the secondary signal salicylic acid as early responses to pathogen attack. We have isolated a soybean cDNA encoding a novel bZIP protein, G/HBF-1, which binds to both the G-box and adjacent H-box in the proximal region of the chalcone synthase chs15 promoter. While G/HBF-1 transcript and protein levels do not increase during the induction of phenylpropanoid biosynthetic genes, G/HBF-1 is phosphorylated rapidly in elicited soybean cells, almost exclusively on serine residues. Using recombinant G/HBF-1 as a substrate, we identified a cytosolic protein-serine kinase that is rapidly and transiently stimulated in cells elicited with either glutathione or an avirulent strain of the soybean pathogen Pseudomonas syringae pv. glycinea. Phosphorylation of G/HBF-1 in vitro enhances binding to the chs15 promoter and we conclude that stimulation of G/HBF-1 kinase activity and G/HBF-1 phosphorylation are terminal events in a signal pathway for activation of early transcription-dependent plant defense responses.

In plants, programed cell death (PCD) is an important mechanism to regulate multiple aspects of growth and development, as well as to remove damaged or infected cells during responses to environmental stresses and pathogen attacks. Under biotic and abiotic stresses, plant cells exhibit a rapid synthesis of nitric oxide (NO) and a parallel accumulation of reactive oxygen species (ROS). Frequently, these responses trigger a PCD process leading to an intrinsic execution of plant cells. The accumulating evidence suggests that both NO and ROS play key roles in PCD. These redox active small molecules can trigger cell death either independently or synergistically. Here we summarize the recent progress on the cross-talk of NO and ROS signals in the hypersensitive response, leaf senescence, and other kinds of plant PCD caused by diverse cues.

We identified rice genes that might be involved in drought stress tolerance by virtue of their anti-apoptotic activity. Potential anti-apoptosis related genes were identified by screening an Oryza sativa cDNA library derived from drought stressed tissues in a yeast functional assay. About 28 O. sativa cDNAs promoted yeast survival following engagement of Bax-induced apoptosis. An O. sativa cDNA encoding R12H780 was a highly conserved putative senescence-associated-protein (OsSAP). OsSAP was both highly and rapidly expressed in response to drought stress. Additionally, OsSAP was found to be localized to the mitochondria. Overall, OsSAP represents a new type of Bax suppressor related gene and endows multiple stress tolerance in yeast.

In plants, the chloroplast is the main reactive oxygen species (ROS) producing site under high light stress. Catalase (CAT), which decomposes hydrogen peroxide (H2 O2 ), is one of the controlling enzymes that maintains leaf redox homeostasis. The catalase mutants with reduced leaf catalase activity from different plant species exhibit an H2 O2 -induced leaf cell death phenotype. This phenotype was differently affected by light intensity or photoperiod, which may be caused by plant species, leaf redox status or growth conditions. In the rice CAT mutant nitric oxide excess 1 (noe1), higher H2 O2 levels induced the generation of nitric oxide (NO) and higher S-nitrosothiol (SNO) levels, suggesting that NO acts as an important endogenous mediator in H2 O2 -induced leaf cell death. As a free radical, NO could also react with other intracellular and extracellular targets and form a series of related molecules, collectively called reactive nitrogen species (RNS). Recent studies have revealed that both RNS and ROS are important partners in plant leaf cell death. Here, we summarize the recent progress on H2 O2 -induced leaf cell death and the crosstalk of RNS and ROS signals in the plant hypersensitive response (HR), leaf senescence, and other forms of leaf cell death triggered by diverse environmental conditions. [Formula: see text] [ Chengcai Chu (Corresponding author)].

Humans have utilised plant derived natural products as medicines for millenia. Moreover, many contemporary pharmaceuticals are also natural products or derivatives thereof. However, the full potential of these compounds remains to be exploited because often they are: complex and difficult to synthesise; found in low quantities; produced by undomesticated and sometimes rare plants; and, their synthesis is routinely influenced by weather conditions. Potentially, the in vitro culture of cells from the corresponding plant species could circumvent some of these problems but the growth of plant cells on an industrial scale is also problematic. The recent isolation and culture of cambial meristematic cells (CMCs), stem cells which ordinarily generate the plant vasculature, may now provide a key platform technology to help realise the full potential of plant natural products.

Nitric oxide (NO) has been proposed to regulate a diverse array of activities during plant growth, development and immune function. S-nitrosylation, the addition of an NO moiety to a reactive cysteine thiol, to form an S-nitrosothiol (SNO), is emerging as a prototypic redox-based post-translational modification. An ARABIDOPSIS THALIANA S-NITROSOGLUTATHIONE (GSNO) REDUCTASE (AtGSNOR1) is thought to be the major regulator of total cellular SNO levels in this plant species. Here, we report on the impact of loss- and gain-of-function mutations in AtGSNOR1 upon plant growth and development. Loss of AtGSNOR1 function in atgsnor1-3 plants increased the number of initiated higher order axillary shoots that remain active, resulting in a loss of apical dominance relative to wild type. In addition atgsnor1-3 affected leaf shape, germination, 2,4-D sensitivity and reduced hypocotyl elongation in both light and dark grown seedlings. Silique size and seed production were also decreased in atgsnor1-3 plants and the latter was reduced in atgsnor1-1 plants, which overexpress AtGSNOR1. Overexpression of AtGSNOR1 slightly delayed flowering time in both long and short days, whereas atgsnor1-3 showed early flowering compared to wild type. In the atgsnor1-3 line, FLOWERING LOCUS C (FLC) expression was reduced, whereas transcription of CONSTANS (CO) was enhanced. Therefore, AtGSNOR1 may negatively regulate the autonomous and photoperiod flowering time pathways. Both overexpression and loss of AtGSNOR1 function also reduced primary root growth, while root hair development was increased in atgsnor1-1 and reduced in atgsnor1-3 plants. Collectively, our findings imply that AtGSNOR1 controls multiple genetic networks integral to plant growth and development.

S-nitrosylation, the covalent attachment of a nitric oxide (NO) moiety to a protein cysteine thiol to form an S-nitrosothiol (SNO) is rapidly emerging as a prototypic, redox-based post-translational modification during plant immune function. Here we review recently identified targets for S-nitrosylation and the consequences of these modifications in relation to the control of plant disease resistance.

Nitric oxide (NO) is a key redox-active, small molecule involved in various aspects of plant growth and development. Here, we report the identification of an NO accumulation mutant, nitric oxide excess1 (noe1), in rice (Oryza sativa), the isolation of the corresponding gene, and the analysis of its role in NO-mediated leaf cell death. Map-based cloning revealed that NOE1 encoded a rice catalase, OsCATC. Furthermore, noe1 resulted in an increase of hydrogen peroxide (H2O2) in the leaves, which consequently promoted NO production via the activation of nitrate reductase. The removal of excess NO reduced cell death in both leaves and suspension cultures derived from noe1 plants, implicating NO as an important endogenous mediator of H2O2-induced leaf cell death. Reduction of intracellular S-nitrosothiol (SNO) levels, generated by overexpression of rice S-nitrosoglutathione reductase gene (GSNOR1), which regulates global levels of protein S-nitrosylation, alleviated leaf cell death in noe1 plants. Thus, S-nitrosylation was also involved in light-dependent leaf cell death in noe1. Utilizing the biotin-switch assay, nanoliquid chromatography, and tandem mass spectrometry, S-nitrosylated proteins were identified in both wild-type and noe1 plants. NO targets identified only in noe1 plants included glyceraldehyde 3-phosphate dehydrogenase and thioredoxin, which have been reported to be involved in S-nitrosylation-regulated cell death in animals. Collectively, our data suggest that both NO and SNOs are important mediators in the process of H2O2-induced leaf cell death in rice.

Multidrug and toxic compound extrusion (MATE) proteins comprise the most recently identified family of multidrug transporters. In plants, the numbers of MATE proteins has undergone a remarkable expansion, underscoring the importance of these transporters within this kingdom.Here, we describe the identification and characterization of Activated Disease Susceptibility 1 (ADS1) which encodes a putative MATE transport protein. An activation tagging screen uncovered the ads1-Dominant (ads1-D) mutant, which was subsequently characterized by molecular, genetic and biochemical approaches.The ads1-D mutant was compromised in both basal and nonhost resistance against microbial pathogens. Further, plant defence responses conferred by RPS4 were also disabled in ads1-D plants. By contrast, depletion of ADS1 transcripts by RNA-interference (RNAi) promoted basal disease resistance. Unexpectedly, ads1-D plants were found to constitutively accumulate reactive oxygen intermediates (ROIs). However, analysis of ads1-D Arabidopsis thaliana respiratory burst oxidase (atrboh) double and triple mutants indicated that an increase in ROIs did not impact ads1-D-mediated disease susceptibility.Our findings imply that ADS1 negatively regulates the accumulation of the plant immune activator salicylic acid (SA) and cognate Pathogenesis-Related 1 (PR1) gene expression. Collectively, these data highlight an important role for MATE proteins in the establishment of plant disease resistance.

A key feature of the plant defence response is the transient engagement of a nitrosative burst, resulting in the synthesis of reactive nitrogen intermediates (RNIs). Specific, highly reactive cysteine (Cys) residues of low pK(a) are a major site of action for these intermediates. The addition of an NO moiety to a Cys thiol to form an S-nitrosothiol (SNO), is termed S-nitrosylation. This redox-based post-translational modification is emerging as a key regulator of protein function in plant immunity. Here we highlight recent advances in our understanding of de-nitrosylation, the mechanism that depletes protein SNOs, with a focus on S-nitrosoglutathione reductase (GSNOR). This enzyme controls total cellular S-nitrosylation indirectly during the defence response by turning over S-nitrosoglutathione (GSNO), a major cache of NO bioactivity.

A plethora of important, chemically diverse natural products are derived from plants. In principle, plant cell culture offers an attractive option for producing many of these compounds. However, it is often not commercially viable because of difficulties associated with culturing dedifferentiated plant cells (DDCs) on an industrial scale. To bypass the dedifferentiation step, we isolated and cultured innately undifferentiated cambial meristematic cells (CMCs). Using a combination of deep sequencing technologies, we identified marker genes and transcriptional programs consistent with a stem cell identity. This notion was further supported by the morphology of CMCs, their hypersensitivity to γ-irradiation and radiomimetic drugs and their ability to differentiate at high frequency. Suspension culture of CMCs derived from Taxus cuspidata, the source of the key anticancer drug, paclitaxel (Taxol), circumvented obstacles routinely associated with the commercial growth of DDCs. These cells may provide a cost-effective and environmentally friendly platform for sustainable production of a variety of important plant natural products.

SUMMARY A complex signal transduction network involving salicylic acid, jasmonic acid and ethylene underlies disease resistance in Arabidopsis. To understand this defence signalling network further, we identified mutants that expressed the marker gene PR-1::luciferase in the absence of pathogen infection. These cir mutants all display constitutive expression of a suite of defence-related genes but exhibit different disease resistance profiles to two biotrophic pathogens, Pseudomonas syringae pv. tomato and Peronospora parasitica NOCO2, and the necrotrophic pathogen Botrytis cinerea. We further characterized cir3, which displays enhanced resistance only to the necrotrophic pathogen. Cir3-mediated resistance to B. cinerea is dependent on accumulated salicylic acid and a functional EIN2 protein.

Changes in redox status are a conspicuous feature of immune responses in a variety of eukaryotes, but the associated signalling mechanisms are not well understood. In plants, attempted microbial infection triggers the rapid synthesis of nitric oxide and a parallel accumulation of reactive oxygen intermediates, the latter generated by NADPH oxidases related to those responsible for the pathogen-activated respiratory burst in phagocytes. Both nitric oxide and reactive oxygen intermediates have been implicated in controlling the hypersensitive response, a programmed execution of plant cells at sites of attempted infection. However, the molecular mechanisms that underpin their function and coordinate their synthesis are unknown. Here we show genetic evidence that increases in cysteine thiols modified using nitric oxide, termed S-nitrosothiols, facilitate the hypersensitive response in the absence of the cell death agonist salicylic acid and the synthesis of reactive oxygen intermediates. Surprisingly, when concentrations of S-nitrosothiols were high, nitric oxide function also governed a negative feedback loop limiting the hypersensitive response, mediated by S-nitrosylation of the NADPH oxidase, AtRBOHD, at Cys 890, abolishing its ability to synthesize reactive oxygen intermediates. Accordingly, mutation of Cys 890 compromised S-nitrosothiol-mediated control of AtRBOHD activity, perturbing the magnitude of cell death development. This cysteine is evolutionarily conserved and specifically S-nitrosylated in both human and fly NADPH oxidase, suggesting that this mechanism may govern immune responses in both plants and animals.

Reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs) have now become well established as important signalling molecules in physiological settings within microorganisms, mammals and plants. These intermediates are routinely synthesised in a highly controlled and transient fashion by NADPH-dependent enzymes, which constitute key regulators of redox signalling. Mild oxidants such as hydrogen peroxide (H(2)O(2)) and especially nitric oxide (NO) signal through chemical reactions with specific atoms of target proteins that result in covalent protein modifications. Specifically, highly reactive cysteine (Cys) residues of low pK(a) are a major site of action for these intermediates. The oxidation of target Cys residues can result in a number of distinct redox-based, post-translational modifications including S-nitrosylation, S-glutathionylation; and sulphenic acid, sulphinic acid and disulphide formation. Importantly, such modifications precisely regulate protein structure and function. Cys-based redox switches are now increasingly being found to underpin many different signalling systems and regulate physiological outputs across kingdoms.

The phenylpropanoid pathway intermediate p-coumaric acid (4-CA) stimulates expression of the bean (Phaseolus vulgaris L.) chalcone synthase (malonyl-CoA:4-coumaroyl-CoA, EC 2.3.1.74) chs15 gene promoter in electroporated protoplasts of alfalfa (Medicago sativa L.). We have analyzed the effects of 5' deletions, mutations, and competition with promoter sequences in trans on the expression of a chs15 promoter-chloramphenicol acetyltransferase gene fusion in elicited alfalfa protoplasts. Two distinct sequence elements, the H-box (consensus CCTACC(N)7CT) and the G-box (CACGTG), are required for stimulation of the chs15 promoter by 4-CA. Furthermore, a 38-base-pair chs15 promoter sequence containing both cis elements conferred responsiveness to 4-CA on the cauliflower mosaic virus 35S minimal promoter. The H-box and G-box in combination establish the complex developmental pattern of chs15 expression and are also involved in stress induction. Hence, potential internal pathway regulation through feed-forward stimulation by 4-CA operates by modulation of the signal pathways for developmental and environmental regulation.

Precise modulation of transcription plays a vital role in both development and the response of all higher organisms to their environment. Temporal activation or repression of specific genes is accomplished via a plethora of transcriptional regulators. However, relatively little is known about how the activities of these proteins are controlled. Recent findings indicate that post-translational modifications fine-tune the function of transcription regulators by affecting their localization, conformation or stability. Here, we discuss these regulatory mechanisms in the context of the plant immune response. This system lends itself particularly well to studies of transcriptional regulators as activation of plant immunity is associated with rapid and dramatic reprogramming of the transcriptome. A case study of the plant immune coactivator NPR1 (nonexpressor of pathogenesis-related (PR) genes 1) illustrates that transcription regulator activity may be controlled by redox-based modifications of cysteine thiols (e.g. disulphide bonding and S-nitrosylation), phosphorylation, and ubiquitinylation coupled to protein degradation. Importantly, cross-talk between distinct protein modifications may determine the spatial and temporal activity of transcription regulators that in turn profile the cellular transcriptome.

Plant disease resistance is one of the most desirable traits in an agricultural context, especially in these times of fear over food security, making it a key topic within plant biology and crop science. This book, which provides an update on a wide variety of important areas within plant pathology, is therefore particularly timely. In the opening chapter, Heath sets the scene and provides a fascinating overview of developments during the last 40 years of plant pathology research from a personal perspective. She places particular emphasis on the hypersensitive response, gene-for-gene resistance and host verses non-host resistance. In concluding, Heath expresses her desire for more cytological and biochemical-based approaches to understand how, in both space and time, given gene products function to establish disease resistance. In the following chapter, Nurnberger and Kemmerling provide an update on pathogen-associated molecular patterns (PAMPs) and PAMP-triggered immunity. Massive strides have been taken in this field in the past few years, which have dramatically increased our appreciation of this defence system, including the isolation of pattern-recognition receptors (PRRs) from potential host plants, the demonstration that PRR function is biologically relevant during the development of disease resistance, and the accumulating evidence that pathogens specifically target this defence mechanism for suppression to aid pathogenesis. Another chapter deals with the type-III secretion system (TTSS) of the bacterial pathogen Pseudomonas syringae. The discovery that bacterial plant pathogens deliver so-called effector proteins directly into the plant cell to aid pathogenesis via this TTSS has primed an explosion of activity in this area over the last few years. Increasing our understanding of the activities and host targets of these effector proteins will likely provide significant insights into exactly how plants repel attempted bacterial infection. Pathogen genomics is another area of plant pathology that has expanded rapidly in recent times. This is particularly true for oomycetes, such as Phytophthora, five species of which have now been sequenced, and the latest advances in this field are discussed in Chapter 5 by Bouwmeester, van Poppel and Govers. Large areas of synteny between these genomes have been found, significantly aiding the gene-calling process. This process can often be difficult because ∼20 % of genes found in these pathogens are thought to be unique to oomycetes. This genomics-based approach identified the RxLR-dEER motif, located adjacent to the signal peptide, which appears to be found in most oomycete effector proteins. This was surprising because no conserved domains have been found to date in fungal avirulence genes. Consequently, candidate oomycete effectors can now be identified rapidly by bioinformatics-based approaches, and their potential function in infection rapidly investigated utilizing high-throughput strategies. The future application of next-generation sequencing approaches such as Roche 454, Illumina-Solexa and Applied Biosystems SOLiD™ will further accelerate the pace of discovery in this research field. Other chapters in the book cover such topics as plant–virus interactions, the role of secondary metabolites at the host–pathogen interface (an old topic that is currently having a renaissance), and lipid signalling in the defence response. A significant strength of this book is that the chapters have been written by experts in their respective fields who have provided up-to-date descriptions of the state-of-the-art. However, perhaps inevitably, there is sometimes redundancy in material between individual contributions. Each chapter is extensively referenced, leading the reader to a potential wealth of further information including research papers and reviews, and the index is extensive. There is generally judicious use of diagrams and tables to complement the text, although some sections would have benefited from more of these. This book is essential reading for final-year undergraduates attending plant pathology courses or undertaking research projects within this area. The collection of updates on key topics also makes this book entirely suitable for graduate students and post-doctoral fellows, or indeed anyone who has a significant interest in this important area of plant science. In sum, this is a well-written and presented book on an exciting and important topic that provides a wealth of up-to-date information suitable for final-year undergraduates, postgraduates and professional researchers.

Cysteine proteases are required for programmed cell death (PCD) in animals. Recent work in Nicotiana benthamiana has implicated cathepsin B-like cysteine proteases in the hypersensitive response (HR) in plants, a form of PCD involved in disease resistance. Here, we investigate the function and regulation of Cathepsin B (CathB) genes in plant defence, and in both pathogen-inducible and developmental forms of PCD. Single, double and triple knockout mutants were isolated for the three Arabidopsis thaliana AtCathB genes. AtCathB genes were redundantly required for full basal resistance against the virulent bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. By contrast, AtCathB genes were not required for R gene-mediated resistance to Pst DC3000 expressing AvrB or AvrRps4. Neither did they contribute to PCD triggered by AvrRps4, although they were crucial for the full development of PCD during HR triggered by AvrB. Cathepsin B has also been proposed to play a positive regulatory role in senescence. Atcathb triple mutants showed a delay in senescence and a seven-fold decrease in accumulation of senescence marker gene SAG12. Our results demonstrate a redundant function for AtCathB genes in basal defence as well as a potential regulatory role in distinct forms of plant PCD.

Calcium (Ca(2+)) transients have been shown to take place in response to diverse developmental and physiological cues. Also, it is involved in biotic and abiotic stress signaling. Nitric oxide (NO) is an important signaling molecule that plays a crucial role in plant growth and development, starting from germination to flowering, ripening of fruit and senescence of organs. Moreover, it plays a pivotal role in several biotic and abiotic stress signaling processes. In the present work, the ability of NO to trigger increases in cytosolic calcium concentration ([Ca(2+)](cyt)) was investigated. For this purpose, transgenic Arabidopsis seedlings constitutively expressing the luminescent Ca(2+)-sensitive protein apoaequorin (35S::APOAEQUORIN) was employed. In chemiluminescence and in vivo Ca(2+) imaging assays, the NO-donor sodium nitroprusside (SNP) triggered a strong, instantaneous, reproducible, and dose-dependent rise in [Ca(2+)](cyt). Moreover, the observed rise in [Ca(2+)](cyt) was shown to be NO-specific and not associated with decomposition products of SNP, as the NO-scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide (C-PTIO) significantly blunted the observed NO-mediated spike in [Ca(2+)](cyt). Interestingly, preincubation of 35S::APOAEQUORIN Arabidopsis seedlings with the plasma membrane channel blocker lanthanum chloride resulted in partial concentration-dependent blocking of the NO-specific Ca(2+) transient. This observation indicates that, in addition to the mobilization of [Ca(2+)](cyt), as an external source in response to NO treatment, there also exists an appreciable contribution of an as yet unidentified internal pool.

The lack of availability of sources of resistance against Alternaria brassicicola within the family Brassicaceae has made oilseed mustard plants a target for one of the most damaging and widespread fungal diseases, Alternaria black spot. Of the other non-host-resistant/tolerant plants, Sinapis alba, white mustard, is considered to be the most important apart from Arabidopsis. To understand the defence response of S. alba upon incompatible interaction with this pathogen, a functional genomic approach using cDNA amplified fragment length polymorphism was performed. The highly reproducible bands, found to be either more amplified or uniquely present in infected S. alba plants compared with non-infected plants, were further subjected to comparative reverse Northern analysis in the incompatible white mustard (S. alba) and compatible India mustard (Brassica juncea L.) plants. The suppression of 46% of the genes in the compatible background indicates the possibility of effective and specific recognition of Alternaria in S. alba. Analysis of the 118 genes up-regulated specifically in infected S. alba compared with B. juncea showed that 98 genes have similarity to proteins such as receptor-like protein kinase genes, genes involved with calcium-mediated signalling and salicylic acid-dependent genes as well as other genes of known function in Arabidopsis. The apparent expression profile data were further confirmed for selected genes by quantitative real-time polymerase chain reaction analysis. Classification of these genes on the basis of their induction pattern in Arabidopsis indicates that the expression profile of several of these genes was distinct in S. alba compared with B. juncea.

Changes in cellular redox status are a well established response across phyla following pathogen challenge. In this context, the synthesis of nitric oxide (NO) is a conspicuous feature of plants responding to attempted microbial infection and this redox-based regulator underpins the development of plant immunity. However, the associated molecular mechanism(s) have not been defined. Here we show that NO accretion during the nitrosative burst promotes increasing S-nitrosylation of the Arabidopsis thaliana salicylic acid-binding protein 3 (AtSABP3) at cysteine (Cys) 280, suppressing both binding of the immune activator, salicylic acid (SA), and the carbonic anhydrase (CA) activity of this protein. The CA function of AtSABP3 is required for the expression of resistance in the host against attempted pathogen infection. Therefore, inhibition of AtSBAP3 CA function by S-nitrosylation could contribute to a negative feedback loop that modulates the plant defense response. Thus, AtSABP3 is one of the first targets for S-nitrosylation in plants for which the biological function of this redox-based post-translational modification has been uncovered. These data provide a molecular connection between the changes in NO levels triggered by attempted pathogen infection and the expression of disease resistance.

A diverse range of plant proteases are implicated in pathogen perception and in subsequent signalling and execution of disease resistance. We demonstrate, using protease inhibitors and virus-induced gene silencing (VIGS), that the plant papain cysteine protease cathepsin B is required for the disease resistance hypersensitive response (HR). VIGS of cathepsin B prevented programmed cell death (PCD) and compromised disease resistance induced by two distinct non-host bacterial pathogens. It also suppressed the HR triggered by transient co-expression of potato R3a and Phytophthora infestans Avr3a genes. However, VIGS of cathepsin B did not compromise HR following recognition of Cladosporium fulvum AVR4 by tomato Cf-4, indicating that plant PCD can be independent of cathepsin B. The non-host HR to Erwinia amylovora was accompanied by a transient increase in cathepsin B transcript level and enzymatic activity and induction of the HR marker gene Hsr203. VIGS of cathepsin B significantly reduced the induction of Hsr203 following E. amylovora challenge, further demonstrating a role for this protease in PCD. Whereas cathepsin B is often relocalized from the lysosome to the cytosol during animal PCD, plant cathepsin B is secreted into the apoplast, and is activated upon secretion in the absence of pathogen challenge.

Salicylic acid (SA) is synthesised by plants in response to challenge by a diverse range of phytopathogens and is essential to the establishment of both local and systemic-acquired resistance (SAR). SA application induces accumulation of pathogenesis-related (PR) proteins. Mutations leading to either reduced SA production or impaired SA perception enhance susceptibility to avirulent and virulent pathogens. However, our knowledge of the primary signalling components activating SA biosynthesis and linking to PR proteins accumulation is rudimentary. We review progress towards characterising key players (NPR1, MPK4) and processes (methylation, amino acid conjugation, S-nitrosylation) contributing to SA-signalling and perception pathways. Further, we examine emerging data on how pathogens have evolved strategies (e.g. ABA modulation and coronatine production) to suppress SA-mediated plant defence.

S-nitrosylation, the covalent attachment of a nitric oxide moiety to a cysteine thiol, is now established as a key post-translational modification in animals. This process has been shown to regulate the function of a wide variety of regulatory, structural, and metabolic proteins. The emerging evidence now suggests that S-nitrosylation may also have a central function in plant biology.

The activated disease resistance (ADR) 1 gene encodes a protein that possesses an N-terminal coiled-coil (CC) motif, nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. ADR1 belongs to a small, atypical Arabidopsis thaliana sub-class containing four CC-NBS-LRR genes. The NBS region of most NBS-LRR proteins possesses numerous conserved motifs. In contrast, the LRR domain, which is subject to positive selection, is highly variable. Surprisingly, sequence analysis revealed that the LRR domain of the ADR1 sub-class was more conserved than the NBS region. Sequence analysis identified two novel conserved motifs, termed TVS and PKAE, specific for this CC-NBS-LRR sub-class. The TVS motif is adjacent to the P-loop, whereas the PKAE motif corresponded to the inter-domain region termed the NBS-LRR linker, which was conserved within the different CC-NBS-LRR classes but varied among classes. These ADR1-specific motifs were employed to identify putative ADR1 homologs in phylogenetically distant and agronomically important plant species. Putative ADR1 homologs were identified in 11 species including rice and in 3 further Poaceae species. The ADR1 sub-class of CC-NBS-LRR proteins is therefore conserved in both monocotyledonous and dicotyledonous plant species.

An activation-tagged allele of activated disease resistance 1 (ADR1) has previously been shown to convey broad spectrum disease resistance. ADR1 was found to encode a coiled-coil (CC)-nucleotide-binding site (NBS)-leucine-rich repeat (LRR) protein, which possessed domains of homology with serine/threonine protein kinases. Here, we show that either constitutive or conditional enhanced expression of ADR1 conferred significant drought tolerance. This was not a general feature of defence-related mutants because cir (constitutive induced resistance)1, cir2 and cpr (constitutive expressor of PR genes)1, which constitutively express systemic acquired resistance (SAR), failed to exhibit this phenotype. Cross-tolerance was not a characteristic of adr1 plants, rather they showed increased sensitivity to thermal and salinity stress. Hence, adr1-activated signalling may antagonise some stress responses. Northern analysis of abiotic marker genes revealed that dehydration-responsive element (DRE)B2A but not DREB1A, RD (response to dehydration)29A or RD22 was expressed in adr1 plant lines. Furthermore, DREB2A expression was salicylic acid (SA) dependent but NPR (non-expressor of PR genes)1 independent. In adr1/ADR1 nahG (naphthalene hydroxylase G), adr1/ADR1 eds (enhanced disease susceptibility)1 and adr1/ADR1 abi1 double mutants, drought tolerance was significantly reduced. Microarray analyses of plants containing a conditional adr1 allele demonstrated that a significant number of the upregulated genes had been previously implicated in responses to dehydration. Therefore, biotic and abiotic signalling pathways may share multiple nodes and their outputs may have significant functional overlap.

Plant immunity against the majority of the microbial pathogens is conveyed by a phenomenon known as non-host resistance (NHR). This defence mechanism affords durable protection to plant species against given species of phytopathogens. We investigated the genetic basis of NHR in Arabidopsis against the wheat powdery mildew fungus Blumeria graminis f. sp. tritici (Bgt). Both primary and appressorial germ tubes were produced from individual Bgt conidia on the surface of the Arabidopsis leaves. Attempted infection occasionally resulted in successful penetration, which led to the development of an abnormal unilateral haustorium. Inoculation of a series of Arabidopsis defence-related mutants with Bgt resulted in the attenuation of reactive oxygen intermediate (ROI) production and salicylic acid (SA)-dependent defence gene expression in eds1, pad4 and nahG plants, which are known to be defective in some aspects of host resistance. Furthermore, Bgt often developed bilateral haustoria in the mutant Arabidopsis lines that closely resembled those formed in wheat. A similar decrease in NHR was observed following treatment of the wild-type Arabidopsis plants with cytochalasin E, an inhibitor of actin microfilament polymerisation. In eds1 mutants, inhibition of actin polymerisation severely compromised NHR in Arabidopsis against Bgt. This permitted completion of the Bgt infection cycle on these plants. Therefore, actin cytoskeletal function and EDS1 activity, in combination, are major contributors to NHR in Arabidopsis against wheat powdery mildew.

A significant limitation of classical loss-of-function screens designed to dissect genetic pathways is that they rarely uncover genes that function redundantly, are compensated by alternative metabolic or regulatory circuits, or which have an additional role in early embryo or gametophyte development. Activation T-DNA tagging is one approach that has emerged in plants to help circumvent these potential problems. This technique utilises a T-DNA sequence that contains four tandem copies of the cauliflower mosaic virus (CaMV) 35S enhancer sequence. This element enhances the expression of neighbouring genes either side of the randomly integrated T-DNA tag, resulting in gain-of-function phenotypes. Activation tagging has identified a number of genes fundamental to plant development, metabolism and disease resistance in Arabidopsis. This review provides selected examples of these discoveries to highlight the utility of this technology. The recent development of activation tagging strategies for other model plant systems and the construction of new more sophisticated vectors for the generation of conditional alleles are also discussed. These recent advances have significantly expanded the horizons for gain-of-function genetics in plants.

Recognition of avirulent microbial pathogens activates an oxidative burst leading to the accumulation of reactive oxygen intermediates (ROIs), which are thought to integrate a diverse set of defence mechanisms resulting in the establishment of plant disease resistance. A novel transgenic Arabidopsis line containing a gst1:luc transgene was developed and employed to report the temporal and spatial dynamics of ROI accumulation and cognate redox signalling in response to attempted infection by avirulent strains of Pseudomonas syringae pv. tomato (Pst). Strong engagement of the oxidative burst was dependent on the presence of functional Pst hrpS and hrpA gene products. Experiments employing pharmacological agents suggested that at least two distinct sources, including an NADPH oxidase and a peroxidase-type enzyme, contributed to the generation of redox cues. The analysis of gst1 and pal1 gene expression in nahG, coi1 and etr1 plants suggested that engagement of the oxidative burst and cognate redox signalling functioned independently of salicylic acid, methyl jasmonate and ethylene. In contrast, studies using a panel of protein kinase and phosphatase inhibitors and in-gel kinase assays in these mutant backgrounds suggested that a 48 kDa mitogen-activated protein kinase (MAPK) activity was required for the activation of gst1 and pal1 in response to redox cues. Thus the engagement of a bifurcating redox signalling pathway possessing a MAPK module may contribute both to the establishment of plant disease resistance, and to the development of cellular protectant mechanisms.

Plants have evolved a plethora of sophisticated defense mechanisms to resist their potential colonization by microbial pathogens and parasites. Preformed physical and chemical barriers constitute the first line of defense. Superimposed upon these is a battery of inducible responses, their engagement

Cauliflower mosaic virus (CaMV) encodes a multifunctional protein P6 that is required for translation of the 35S RNA and also acts as a suppressor of RNA silencing. Here we demonstrate that P6 additionally acts as a pathogenicity effector of an unique and novel type, modifying NPR1 (a key regulator of salicylic acid (SA)- and jasmonic acid (JA)-dependent signaling) and inhibiting SA-dependent defence responses We find that that transgene-mediated expression of P6 in Arabidopsis and transient expression in Nicotiana benthamiana has profound effects on defence signaling, suppressing expression of representative SA-responsive genes and increasing expression of representative JA-responsive genes. Relative to wild-type Arabidopsis P6-expressing transgenics had greatly reduced expression of PR-1 following SA-treatment, infection by CaMV or inoculation with an avirulent bacterial pathogen Pseudomonas syringae pv tomato (Pst). Similarly transient expression in Nicotiana benthamiana of P6 (including a mutant form defective in translational transactivation activity) suppressed PR-1a transcript accumulation in response to Agrobacterium infiltration and following SA-treatment. As well as suppressing the expression of representative SA-regulated genes, P6-transgenic Arabidopsis showed greatly enhanced susceptibility to both virulent and avirulent Pst (titres elevated 10 to 30-fold compared to non-transgenic controls) but reduced susceptibility to the necrotrophic fungus Botrytis cinerea. Necrosis following SA-treatment or inoculation with avirulent Pst was reduced and delayed in P6-transgenics. NPR1 an important regulator of SA/JA crosstalk, was more highly expressed in the presence of P6 and introduction of the P6 transgene into a transgenic line expressing an NPR1:GFP fusion resulted in greatly increased fluorescence in nuclei even in the absence of SA. Thus in the presence of P6 an inactive form of NPR1 is mislocalized in the nucleus even in uninduced plants. These results demonstrate that P6 is a new type of pathogenicity effector protein that enhances susceptibility to biotrophic pathogens by suppressing SA-but enhancing JA-signaling responses.

Candida albicans is a major fungal pathogen that causes frequent mucosal infections in otherwise healthy individuals, and life-threatening systemic infections in immune-compromised patients. The first line of defence against Candida albicans is the release of reactive nitrogen species (RNS), reactive oxygen species (ROS) and cationic fluxes by macrophages and neutrophils. As a result robust responses to RNS, ROS and cationic stresses play major roles in the virulence of C. albicans . This fungus is exposed to all of these stresses simultaneously following phagocytosis. Consequently in this study we examined the responses of this pathogen to combinations of nitrosative, oxidative and cationic stress. The roles of the Cta4-Yhb1, Cap1, Hog1 and Mkc1 signalling pathways, which activate responses to individual nitrosative, oxidative, cationic and cell wall stresses, respectively, were examined. The sensitivity of null mutants to individual and combinatorial stresses was examined, and the activation of these pathways was confirmed by assaying the phosphorylation levels of the key protein kinases. Further evidence of the pathway activation was provided by transcription factor localisation and the expression of downstream target genes. We show that combinatorial nitrosative, oxidative and/or cationic stresses exert unexpected effects upon the classical stress signalling pathways. The relevance to C. albicans infections will be discussed.

In legumes, isoflavonoid derivatives function as antimicrobial phytoalexins, whereas phytoalexins of solanaceous species are of terpenoid origin. The phenylpropanoid and isoprenoid pathways leading to these phytoalexins are involved in the synthesis of a wide range of secondary metabolites with important functions in plant growth, development and responses to the environment. Elicitation of phytoalexin biosynthesis involves transcriptional activation of the genes encoding enzymes of general phenylpropanoid/terpenoid biosynthesis, and of the genes for the specific branch pathways leading to antimicrobial compounds. In order to understand the molecular controls determining the developmental and environmental regulation of the general and specific enzymes of phytoalexin synthesis, we are studying the promoter regions of three elicitor inducible genes, chalcone synthase (chs, isoflavonoid pathway, general), isoflavone reductase (ifr,isoflavonoid pathway, specific) and 3-hydroxy-3-methylglutaryl CoA reductase (hmgr,terpenoid pathway, general). We describe cis-elements and trans-factors involved in the expression of these genes in relation to their tissue specific expression and response to biotic stress. Two elements, the G-box and H-box, located within 50 bp of the TATA box, are important for regulation of expression of chs and probably hmgr, but are not present in the alfalfa ifr promoter.

Alfalfa (Medicago sativa L.) is probably the world’s most important forage crop. As a nitrogen-fixing legume, it is of value as a source of reduced nitrogen, and is becoming increasingly popular as a soil-conserving perennial in low-input sustainable agricultural systems. It is a cross-pollinated autotetraploid which, being highly heterozygous, exhibits substantial genetic diversity between individual plants of a particular cultivar. The complexity of alfalfa genetics is a hindrance to any form of molecular investigation which involves genetic crosses; however, alfalfa is amenable to stable genetic transformation with Agro-bacterium tumefaciens or A. rhizogenes, 1-6 and is readily, if slowly, regenerable from culture via somatic embryogenesis.7-9 It is therefore a suitable target for improvement by recombinant DNA technology.

A chimeric gene construct containing a bean chalcone synthase (CHS) promoter fused to the chloramphenicol acetyltransferase (CAT) reporter gene was strongly expressed when electroporated into alfalfa protoplasts that were then exposed to a fungal elicitor. Low concentrations (5 x 10(-6) to 10(-4) M) of exogenously applied trans-cinnamic acid (CA), the first intermediate of the phenylpropanoid pathway, slightly stimulated elicitor-induced CAT expression, whereas high concentrations (greater than 10(-4) M) severely reduced expression to below the levels observed in the absence of elicitor. In contrast, trans-p-coumaric acid (4-CA, the second intermediate in the pathway) stimulated expression from the CHS promoter up to 4.5-fold at 5 x 10(-4) M. Expression of CAT driven by the promoters of other elicitor-inducible defense response genes was not markedly affected by CA or 4-CA. Stimulation of CHS promoter expression by low concentrations of CA and 4-CA was completely abolished by 59 deletion to position -130, but not -174. When the -180 to -130 region of the CHS15 promoter was coelectroporated into elicited protoplasts on a separate plasmid along with the intact -326 CHS-CAT construct, the decreased CAT expression as a function of CA or 4-CA concentration was consistent with the coelectroporated sequence competing in trans with the intact promoter for the binding of a factor(s) involved in the up regulation of CHS transcription by 4-CA and low concentrations of CA. Our data support the hypothesis that phenylpropanoid compounds may act as natural and specific regulators of plant gene expression and define the location of a cis-acting element in the CHS15 promoter involved in the induction by phenylpropanoid pathway intermediates.

Twelve phenolic compounds with related structures were analyzed for their ability to act as chemoattractants for Agrobacterium tumefaciens C58C1 and as inducers of the Ti plasmid virulence operons. The results divided the phenolic compounds into three groups: compounds that act as strong vir inducers and are chemoattractants for A. tumefaciens C58C1 harboring the nopaline Ti plasmid pDUB1003 delta 31, but not the isogenic cured strain; compounds that are at best weak vir inducers and are weak chemoattractants for Ti plasmid-harboring and cured A. tumefaciens C58C1; and compounds that are vir noninducers and are also nonattractants. A strong correlation between vir-inducing ability and Ti plasmid requirement for chemotaxis is thus established. In addition, chemical structure rules for vir induction and chemotaxis are outlined. Positive chemotaxis toward root and shoot homogenates from monocotyledonous and dicotyledonous plants was observed. At low extract concentrations, chemotaxis was enhanced by the presence of Ti plasmid. The chemoattractants do not derive from intact cell walls. Lack of attraction is not responsible for the apparent block to monocot transformation by A. tumefaciens.

Summary: Chemotaxis of Rhizobium leguminosarum biovar phaseoli RP8002 towards a range of carbohydrates, phenolic compounds and flavonoids was assayed. Xylose (peak response 10−4 M), sucrose (peak response 10−6 M) and raffinose (peak response 10−5 M) were strong chemoattractants amongst the carbohydrates, whilst glucose, fructose, galactose and maltose produced little or no detectable response. Of the monocyclic phenolic compounds, vanillyl alcohol, p-hydroxybenzoic acid (both peak responses 10−6 M) and 3,4-dihydroxybenzoic acid (peak response 10−4 M) all evoked strong chemotactic responses. Amongst the nod-inducing flavonoids, apigenin and luteolin were both strong chemoattractants (peaks at 10−5 M) while naringenin produced a very low response. Competition experiments suggest that apigenin and luteolin are recognized by a common receptor, but that there exists a separate receptor for luteolin alone. The inhibitors of nod-induction, umbelliferone and acetosyringone, both produced strong chemotactic responses, with peaks at 10−3 M and 10−2 M respectively. This evidence is indicative of a role for chemotaxis towards nod-inducing flavonoids in the initiation of root nodule formation by rhizobia, and also suggests that chemotaxis may influence the host range of the interaction.

Summary: Motility of Agrobacterium tumefaciens C58C1 consisted of long straight runs, with relatively few tumbles. Speeds of up to 60 μm s−1 and runs of up to 500 μm were recorded. The propulsive mechanism appeared to resemble that of Rhizobium. Chemotaxis towards carbohydrates resolved four groups of sugars: chemoattractants with peaks at 10−6 M (sucrose, glucose and fructose); 10−5 M (maltose, lactulose and galactose); 10−4 M (raffinose, stachyose and arabinose); and weak or non-attractants (palatinose, lactose, cellobiose and xylose). In descending order, the magnitude of the responses was as follows: sucrose ≫ maltose > lactulose > glucose > galactose/fructose > stachyose/arabinose/raffinose. The amino acids valine and arginine were good chemoattractants with peaks at 10−3 M, but no significant attraction was observed with alanine, cysteine, methionine or glycine. These results are indicative of a highly sensitive chemotaxis system towards sugars in A. tumefaciens C58C1, and suggest a role for this process in the ecology of the organism.

Our interest in Chemotaxis stems from the concept that unless bacteria are present in huge numbers, and evenly distributed throughout the soil, they must arrive from somewhere to interact with a plant. Agrobaeterium tumefaeiens thus possesses a multifunctional system capable of triggering either Chemotaxis or gene-induction depending upon the ligand concentration, as part of an intricate mechanism for sensing and guiding A. tumefaeiens towards wounded cells.

Agrobacterium tumefaciens exhibits an unusual flagellation pattern in the electron microscope. The typical pattern is of two flagella in a polar or sub-polar tuft at one pole only, plus one or two lateral flagella on each side. Twenty independent behavioural mutants were isolated, after Tn5 mutagenesis, and divided into several classes. Fourteen non-motile mutants were differentiable into non-flagellate and flagellate subclasses, one non-chemotactic mutant appeared to have a defect in the intracellular signalling pathway, and one other mutant displayed an unusual pattern of polar cell pairing. Four mutants with impaired swarming ability were also isolated. Root colonization ability was impaired in the non-motile mutant for which this property was tested. From all but three of the mutants, the Tn5 insertion sites, including flanking sequences, have been cloned and used as probes to isolate a set of seven cosmids from a wild-type A. tumefaciens library. No hybridization to Escherichia coli or Pseudomonas DNA was detected for the A. tumefaciens mutant flanking sequences, but varying degrees of similarity to Rhizobium meliloti behavioural genes were detected. By complementation of A. tumefaciens behavioural mutants and hybridization of mutant flanking sequences, one cosmid insert was shown to contain at least nine behavioural loci, and another, linked to it on the chromosome, at least three. This suggests that, as in other organisms, behavioural genes are clustered in the A. tumefaciens genome.