Amino acid homeostasis modulates salicylic acid-associated redox status and defense responses in Arabidopsis

The tight association between nitrogen status and pathogenesis has been broadly documented in plant-pathogen interactions. However, the interface between primary metabolism and disease responses remains largely unclear. Here, we show that knockout of a single amino acid transporter, LYSINE HISTIDINE TRANSPORTER1 (LHT1), is sufficient for Arabidopsis thaliana plants to confer a broad spectrum of disease resistance in a salicylic acid-dependent manner. We found that redox fine-tuning in photosynthetic cells was causally linked to the lht1 mutant-associated phenotypes. Furthermore, the enhanced resistance in lht1 could be attributed to a specific deficiency of its main physiological substrate, Gln, and not to a general nitrogen deficiency. Thus, by enabling nitrogen metabolism to moderate the cellular redox status, a plant primary metabolite, Gln, plays a crucial role in plant disease resistance.     

Arabidopsis thaliana EDS4 contributes to salicylic acid (SA)-dependent expression of defense responses: evidence for inhibition of jasmonic acid signaling by SA

The Arabidopsis enhanced disease susceptibility 4 (eds4) mutation causes enhanced susceptibility to infection by the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326). Gene-for-gene resistance to bacteria carrying the avirulence gene avrRpt2 is not significantly affected by eds4. Plants homozygous for eds4 exhibit reduced expression of the pathogenesis-related gene PR-1 after infection by Psm ES4326, weakened responses to treatment with the signal molecule salicylic acid (SA), impairment of the systemic acquired resistance response, and reduced accumulation of SA after infection with Psm ES4326. These phenotypes indicate that EDS4 plays a role in SA-dependent signaling. SA has been shown to have a negative effect on activation of gene expression by the signal molecule jasmonic acid (JA). Two mutations that cause reduced SA levels, eds4 and pad4, cause heightened responses to inducers of JA-dependent gene expression, providing genetic evidence to support the idea that SA interferes with JA-dependent signaling. Two possible working models of the role of EDS4 in governing activation of defense responses are presented.     

Arabidopsis local resistance to Botrytis cinerea involves salicylic acid and camalexin and requires EDS4 and PAD2, but not SID2, EDS5 or PAD4

Salicylic acid (SA) is an important regulator of plant defense responses, and a variety of Arabidopsis mutants impaired in resistance against bacterial and fungal pathogens show defects in SA accumulation, perception, or signal transduction. Nevertheless, the role of SA-dependent defense responses against necrotrophic fungi is currently unclear. We determined the susceptibility of a set of previously identified Arabidopsis mutants impaired in defense responses to the necrotrophic fungal pathogen Botrytis cinerea. The rate of development of B. cinerea disease symptoms on primary infected leaves was affected by responses mediated by the genes EIN2, JAR1, EDS4, PAD2, and PAD3, but was largely independent of EDS5, SID2/ICS1, and PAD4. Furthermore, plants expressing a nahG transgene or treated with a phenylalanine ammonia lyase (PAL) inhibitor showed enhanced symptoms, suggesting that SA synthesized via PAL, and not via isochorismate synthase (ICS), mediates lesion development. In addition, the degree of lesion development did not correlate with defensin or PR1 expression, although it was partially dependent upon camalexin accumulation. Although npr1 mutant leaves were normally susceptible to B. cinerea infection, a double ein2 npr1 mutant was significantly more susceptible than ein2 plants, and exogenous application of SA decreased B. cinerea lesion size through an NPR1-dependent mechanism that could be mimicked by the cpr1 mutation. These data indicate that local resistance to B. cinerea requires ethylene-, jasmonate-, and SA-mediated signaling, that the SA affecting this resistance does not require ICS1 and is likely synthesized via PAL, and that camalexin limits lesion development.     

Discrimination of Arabidopsis PAD4 activities in defense against green peach aphid and pathogens

The Arabidopsis (Arabidopsis thaliana) lipase-like protein PHYTOALEXIN DEFICIENT4 (PAD4) is essential for defense against green peach aphid (GPA; Myzus persicae) and the pathogens Pseudomonas syringae and Hyaloperonospora arabidopsidis. In basal resistance to virulent strains of P. syringae and H. arabidopsidis, PAD4 functions together with its interacting partner ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) to promote salicylic acid (SA)-dependent and SA-independent defenses. By contrast, dissociated forms of PAD4 and EDS1 signal effector-triggered immunity to avirulent strains of these pathogens. PAD4-controlled defense against GPA requires neither EDS1 nor SA. Here, we show that resistance to GPA is unaltered in an eds1 salicylic acid induction deficient2 (sid2) double mutant, indicating that redundancy between EDS1 and SID2-dependent SA, previously reported for effector-triggered immunity conditioned by certain nucleotide-binding-leucine-rich repeat receptors, does not explain the dispensability of EDS1 and SID2 in defense against GPA. Mutation of a conserved serine (S118) in the predicted lipase catalytic triad of PAD4 abolished PAD4-conditioned antibiosis and deterrence against GPA feeding, but S118 was dispensable for deterring GPA settling and promoting senescence in GPA-infested plants as well as for pathogen resistance. These results highlight distinct molecular activities of PAD4 determining particular aspects of defense against aphids and pathogens.     

The salicylic acid loop in plant defense

Salicylic acid is an important signal molecule in plant defense. In the past two years, significant progress has been made in understanding the mechanism of salicylic-acid biosynthesis and signaling in plants. A pathway similar to that found in some bacteria synthesizes salicylic acid from chorismate via isochorismate. Salicylic-acid signaling is mediated by at least two mechanisms, one requiring the NON-EXPRESSOR OF PR1 (NPR1) gene and a second that is independent of NPR1. Feedback loops involving salicylic acid modulate upstream signals. These feedback loops may provide a point for integrating developmental, environmental and other defense-associated signals, and thus fine-tune the defense responses of plants.     

The GH3 acyl adenylase family member PBS3 regulates salicylic acid-dependent defense responses in Arabidopsis

The pbs3-1 mutant, identified in a screen for Arabidopsis (Arabidopsis thaliana) mutants exhibiting enhanced susceptibility to the avirulent Pseudomonas syringae pathogen DC3000 (avrPphB), also exhibits enhanced susceptibility to virulent P. syringae strains, suggesting it may impact basal disease resistance. Because induced salicylic acid (SA) is a critical mediator of basal resistance responses, free and glucose-conjugated SA levels were measured and expression of the SA-dependent pathogenesis-related (PR) marker, PR1, was assessed. Surprisingly, whereas accumulation of the SA glucoside and expression of PR1 were dramatically reduced in the pbs3-1 mutant in response to P. syringae (avrRpt2) infection, free SA was elevated. However, in response to exogenous SA, the conversion of free SA to SA glucoside and the induced expression of PR1 were similar in pbs3-1 and wild-type plants. Through positional cloning, complementation, and sequencing, we determined that the pbs3-1 mutant contains two point mutations in the C-terminal region of the protein encoded by At5g13320, resulting in nonconserved amino acid changes in highly conserved residues. Additional analyses with Arabidopsis containing T-DNA insertion (pbs3-2) and transposon insertion (pbs3-3) mutations in At5g13320 confirmed our findings with pbs3-1. PBS3 (also referred to as GH3.12) is a member of the GH3 family of acyl-adenylate/thioester-forming enzymes. Characterized GH3 family members, such as JAR1, act as phytohormone-amino acid synthetases. Thus, our results suggest that amino acid conjugation plays a critical role in SA metabolism and induced defense responses, with PBS3 acting upstream of SA, directly on SA, or on a competitive inhibitor of SA.     

Genes controlling expression of defense responses in Arabidopsis.

In the past year, two regulatory defense-related genes, EDS1l and COl1, have been cloned. Several other genes with regulatory functions have been identified by mutation, including DND1, PAD4, CPR6, and SSl1. It has become clear that jasmonate signaling plays an important role in defense response signaling, and that the jasmonate and salicylic acid signaling pathways are interconnected.     

Genetic dissection of salicylic acid-mediated defense signaling networks in Arabidopsis

Properly coordinated defense signaling networks are critical for the fitness of plants. One hub of the defense networks is centered on salicylic acid (SA), which plays a key role in activating disease resistance in plants. However, while a number of genes are known to affect SA-mediated defense, relatively little is known about how these gene interact genetically with each other. Here we exploited the unique defense-sensitized Arabidopsis mutant accelerated cell death (acd) 6-1 to dissect functional relationships among key components in the SA hub. We show that while enhanced disease susceptibility (eds) 1-2 and phytoalexin deficient (pad) 4-1 suppressed acd6-1-conferred small size, cell death, and defense phenotypes, a combination of these two mutations did not incur additive suppression. This suggests that EDS1 and PAD4 act in the same signaling pathway. To further evaluate genetic interactions among SA regulators, we constructed 10 pairwise crosses in the acd6-1 background among mutants defective in: SA INDUCTION-DEFICIENT 2 for SA biosynthesis; AGD2-LIKE DEFENSE 1, EDS5, and PAD4 for SA accumulation; and NONEXPRESSOR OF PR GENES 1 for SA signaling. Systematic analysis of the triple mutants based on their suppression of acd6-1-conferred phenotypes revealed complex and interactive genetic relationships among the tested SA genes. Our results suggest a more comprehensive view of the gene networks governing SA function and provide a framework for further interrogation of the important roles of SA and possibly other signaling molecules in regulating plant disease resistance.     

Arabidopsis GH3-LIKE DEFENSE GENE 1 is required for accumulation of salicylic acid, activation of defense responses and resistance to Pseudomonas syringae

In Arabidopsis, the GH3-like gene family consists of 19 members, several of which have been shown to adenylate the plant hormones jasmonic acid, indole acetic acid and salicylic acid (SA). In some cases, this adenylation has been shown to catalyze hormone conjugation to amino acids. Here we report molecular characterization of the GH3-LIKE DEFENSE GENE 1 (GDG1), a member of the GH3-like gene family, and show that GDG1 is an important component of SA-mediated defense against the bacterial pathogen Pseudomonas syringae. Expression of GDG1 is induced earlier and to a higher level in response to avirulent pathogens compared to virulent pathogens. gdg1 null mutants are compromised in several pathogen defense responses, including activation of defense genes and resistance against virulent and avirulent bacterial pathogens. Accumulation of free and glucoside-conjugated SA (SAG) in response to pathogen infection is compromised in gdg1 mutants. All defense-related phenotypes of gdg1 can be rescued by external application of SA, suggesting that gdg1 mutants are defective in the SA-mediated defense pathway(s) and that GDG1 functions upstream of SA. Our results suggest that GDG1 contributes to both basal and resistance gene-mediated inducible defenses against P. syringae (and possibly other pathogens) by playing a critical role in regulating the levels of pathogen-inducible SA. GDG1 is allelic to the PBS3 (avrPphB susceptible) gene.     

Cross-talk between gibberellins and salicylic acid in early stress responses in Arabidopsis thaliana seeds

Salicylic acid (SA) is a plant hormone mainly associated with the induction of defense mechanism in plants, although in the last years there is increasing evidence on the role of SA in plant responses to abiotic stress. We recently reported that an increase in endogenous SA levels are able to counteract the inhibitory effects of several abiotic stress conditions during germination and seedling establishment of Arabidopsis thaliana and that this effect is modulated by gibberellins (GAs) probably through a member of the GASA (Giberellic Acid Stimulated in Arabidopsis) gene family, clearly showing the existence of a cross talk between these two plant hormones in Arabidopsis.Key words: abiotic stress responses, Arabidopsis thaliana, gibberellins, hormone cross-talk, salicylic acidGAs and SA play important roles in many processes of plant growth and development, and despite the recent papers reporting the existence of a complex network of hormone interactions, evidences of a cross talk between these two plant hormones have been very scarce.^1,2 These authors indicate that GAs are able to regulate SA biosynthesis during plant responses to pathogens. Interestingly, ABA has recently been proved to negative regulate SA-mediated defenses by downregulating SA biosynthesis.³ These data are consistent with the well known ABA/GAs antagonistic regulation of many aspects of plant development, such as seed dormancy or germination.^4,5 Thus, it seems clear that ABA and GAs are able to control plant immune responses by modulating the levels of salicylic acid and/or jasmonic acid.^1–3 In addition to the role of GAs in the regulation of plant responses to biotic stress, we have recently documented a role of GAs in early plant abiotic stress responses in Arabidopsis through modulation of SA levels,⁶ hormone that been involved in responses to abiotic stress conditions.⁷ For instance, it has been proved that SA has an important role in heat stress responses⁸ or in the improved germination of Arabidopsis thaliana seeds under salt stress conditions.⁹We showed that GAs and the overexpression of a GA-responsive gene were able to increase not only endogenous levels of SA, but also the expression of ics1 and npr1 genes, involved in SA biosynthesis and action, respectively.⁶ In addition, we have also analyzed expression levels of other genes that have been reported as SA-regulated. For instance, isocitrate lyase, a key enzyme involved in lipid metabolism during seed germination¹⁰ and a good marker of seed vigor under stress conditions,¹¹ was found to be induced by SA in germinated seeds of Arabidopsis thaliana.⁹ Thus, we proved that the expression of isocitrate lyase was upregulated in GASA4 overexpressing lines, and after exogenous application of GA₃ (Fig. 1), both situations increasing endogenous SA levels.⁶ We have documented that SA may have a role in some of the physiological processes associated with GAs, since exogenous application of SA was able to both revert the inhibitory effect of PCB on seed germination and improve germination of the GA-deficient mutant ga1–3.⁶ Thus, we can hypothesize that the GA-mediated induction of isocitrate lyase gene observed in Arabidopsis thaliana is the result of the increased levels of SA detected either after overexpression of the GA-induced GASA4 gene in Arabidopsis or after exogenous application of gibberellic acid. In other words, GAs are able to induce the expression of isocitrate lyase gene in a SA-dependent manner, producing the establishment of a vigorous seedling.⁹ These data support the idea that GAs may have an important role in SA biosynthesis and action, and that some of the physiological effects of this hormone may be mediate by SA. In summary, our results clearly show the existence of a cross talk between these two plant hormones during Arabidopsis thaliana seeds germination and early seedling growth under abiotic stress conditions, showing another junction in the complex mechanism of hormone interactions.Open in a separate windowFigure 1(A) Expression of the isocitrate lyase gene in FsGASA-overexpressing plants (G1 to G3) compared to Col-0. (B) Expression of the isocitrate lyase gene in Arabidopsis seedlings treated or not with 100 µM GA₃. mRNA levels were determined by northern blot analysis using total RNAs (10 µg/line) isolated from 7 d-old seedlings. Bottom, ethidium bromide stained gels showing rRNAs. Top: quantification of hybridization signals obtained by using a phosphoimage scanner. Data were normalized to the rRNA value. Blots were repeated twice and yielded similar results.     

The Arabidopsis NPR1 protein is a receptor for the plant defense hormone salicylic acid

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Highlights? NPR1 is a salicylic acid (SA) receptor, binding specifically to SA via Cys^521/529 ? NPR1 binds copper in vivo via Cys^521/529, and metals are required for SA binding ? SA directly regulates the conformation of NPR1 by deoligomerizing NPR1 into a dimer ? The NPR1 BTB/POZ domain autoinhibits the function of the NPR1 transactivation domain     

Evidence that the role of plant defensins in radish defense responses is independent of salicylic acid

Radish leaves contain two homologous 5-kDa plant defensins which accumulate systemically upon infection by fungal pathogens (F.R.G. Terras et al., 1995, Plant Cell 7: 573–588). Here we report on the molecular cloning of the cDNAs encoding the two pathogen-inducible plant defensin isoforms from radish (Raphanus sativus L.) leaves. Tissue-print and whole-leaf electroblot immunostaining showed that the plant defensin peptides not only accumulate at high levels at or immediately around the infection sites in leaves inoculated with Alternariabrassicicola, but also accumulate in healthy tissue further away from the infection sites and in non-infected leaves from infected plants. Gel blot analysis of RNA confirmed that expression of plant defensin genes is systemically triggered upon fungal infection whereas radish PR-1 gene expression is only activated locally. In contrast to the radish PR-1 gene(s), expression of the radish plant defensin genes was not induced by external application of salicylic acid. Activation of the plant defensin genes, but not that of PR-1 genes, occurred upon treatment with methyl jasmonate, ethylene and paraquat. Received: 3 December 1997 / Accepted: 3 March 1998