Antony Buchala

Concomitant Activation of Jasmonate and Ethylene Response Pathways Is Required for Induction of a Plant Defensin Gene in Arabidopsis

Activation of the plant defensin gene PDF1.2 in Arabidopsis by pathogens has been shown previously to be blocked in the ethylene response mutant ein2-1 and the jasmonate response mutant coi1-1. In this work, we have further investigated the interactions between the ethylene and jasmonate signal pathways for the induction of this defense response. Inoculation of wild-type Arabidopsis plants with the fungus Alternaria brassicicola led to a marked increase in production of jasmonic acid, and this response was not blocked in the ein2-1 mutant. Likewise, A. brassicicola infection caused stimulated emission of ethylene both in wild-type plants and in coi1-1 mutants. However, treatment of either ein2-1 or coi1-1 mutants with methyl jasmonate or ethylene did not induce PDF1.2, as it did in wild-type plants. We conclude from these experiments that both the ethylene and jasmonate signaling pathways need to be triggered concomitantly, and not sequentially, to activate PDF1.2 upon pathogen infection. In support of this idea, we observed a marked synergy between ethylene and methyl jasmonate for the induction of PDF1.2 in plants grown under sterile conditions. In contrast to the clear interdependence of the ethylene and jasmonate pathways for pathogen-induced activation of PDF1.2, functional ethylene and jasmonate signaling pathways are not required for growth responses induced by jasmonate and ethylene, respectively.

NPR1 Modulates Cross-Talk between Salicylate- and Jasmonate-Dependent Defense Pathways through a Novel Function in the Cytosol

Plant defenses against pathogens and insects are regulated differentially by cross-communicating signal transduction pathways in which salicylic acid (SA) and jasmonic acid (JA) play key roles. In this study, we investigated the molecular mechanism of the antagonistic effect of SA on JA signaling. Arabidopsis plants unable to accumulate SA produced 25-fold higher levels of JA and showed enhanced expression of the JA-responsive genes LOX2, PDF1.2, and VSP in response to infection by Pseudomonas syringae pv tomato DC3000, indicating that in wild-type plants, pathogen-induced SA accumulation is associated with the suppression of JA signaling. Analysis of the Arabidopsis mutant npr1, which is impaired in SA signal transduction, revealed that the antagonistic effect of SA on JA signaling requires the regulatory protein NPR1. Nuclear localization of NPR1, which is essential for SA-mediated defense gene expression, is not required for the suppression of JA signaling, indicating that cross-talk between SA and JA is modulated through a novel function of NPR1 in the cytosol.

Signal Signature and Transcriptome Changes of Arabidopsis During Pathogen and Insect Attack

Plant defenses against pathogens and insects are regulated differentially by cross-communicating signaling pathways in which salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) play key roles. To understand how plants integrate pathogen- and insect-induced signals into specific defense responses, we monitored the dynamics of SA, JA, and ET signaling in Arabidopsis after attack by a set of microbial pathogens and herbivorous insects with different modes of attack. Arabidopsis plants were exposed to a pathogenic leaf bacterium (Pseudomonas syringae pv. tomato), a pathogenic leaf fungus (Alternaria brassicicola), tissue-chewing caterpillars (Pieris rapae), cell-content-feeding thrips (Frankliniella occidentalis), or phloem-feeding aphids (Myzus persicae). Monitoring the signal signature in each plant-attacker combination showed that the kinetics of SA, JA, and ET production varies greatly in both quantity and timing. Analysis of global gene expression profiles demonstrated that the signal signature characteristic of each Arabidopsis-attacker combination is orchestrated into a surprisingly complex set of transcriptional alterations in which, in all cases, stress-related genes are overrepresented. Comparison of the transcript profiles revealed that consistent changes induced by pathogens and insects with very different modes of attack can show considerable overlap. Of all consistent changes induced by A. brassicicola, Pieris rapae, and E occidentalis, more than 50% also were induced consistently by P. syringae. Notably, although these four attackers all stimulated JA biosynthesis, the majority of the changes in JA-responsive gene expression were attacker specific. All together, our study shows that SA, JA, and ET play a primary role in the orchestration of the plants defense response, but other regulatory mechanisms, such as pathway cross-talk or additional attacker-induced signals, eventually shape the highly complex attacker-specific defense response.

Induced Systemic Resistance in Arabidopsis thaliana in Response to Root Inoculation with Pseudomonas fluorescens CHA0

Root inoculation of Arabidopsis thaliana ecotype Columbia with Pseudomonas fluorescens CHA0r partially protected leaves from the oomycete Peronospora parasitica. The molecular determinants of Pseudomonas fluorescens CHA0r for this induced systemic resistance (ISR) were investigated, using mutants derived from strain CHA0: CHA400 (pyoverdine deficient), CHA805 (exoprotease deficient), CHA77 (HCN deficient), CHA660 (pyoluteorin deficient), CHA631 (2,4-diacetylphloroglucinol [DAPG] deficient), and CHA89 (HCN, DAPG- and pyoluteorin deficient). Only mutations interfering with DAPG production led to a significant decrease in ISR to Peronospora parasitica. Thus, DAPG production in Pseudomonas fluorescens is required for the induction of ISR to Peronospora parasitica. DAPG is known for its antibiotic activity; however, our data indicate that one action of DAPG could be due to an effect on the physiology of the plant. DAPG at 10 to 100 microM applied to roots of Arabidopsis mimicked the ISR effect. CHA0r-mediated ISR was also tested in various Arabidopsis mutants and transgenic plants: NahG (transgenic line degrading salicylic acid [SA]), sid2-1 (nonproducing SA), npr1-1 (non-expressing NPR1 protein), jar1-1 (insensitive to jasmonic acid and methyl jasmonic acid), ein2-1 (insensitive to ethylene), etr1-1 (insensitive to ethylene), eir1-1 (insensitive to ethylene in roots), and pad2-1 (phytoalexin deficient). Only jar1-1, eir1-1, and npr1-1 mutants were unable to undergo ISR. Sensitivity to jasmonic acid and functional NPR1 and EIR1 proteins were required for full expression of CHA0r-mediated ISR. The requirements for ISR observed in this study in Peronospora parasitica induced by Pseudomonas fluorescens CHA0r only partially overlap with those published so far for Peronospora parasitica, indicating a great degree of flexibility in the molecular processes leading to ISR.

The PP2C-Type Phosphatase AP2C1, Which Negatively Regulates MPK4 and MPK6, Modulates Innate Immunity, Jasmonic Acid, and Ethylene Levels in Arabidopsis

Wound signaling pathways in plants are mediated by mitogen-activated protein kinases (MAPKs) and stress hormones, such as ethylene and jasmonates. In Arabidopsis thaliana, the transmission of wound signals by MAPKs has been the subject of detailed investigations; however, the involvement of specific phosphatases in wound signaling is not known. Here, we show that AP2C1, an Arabidopsis Ser/Thr phosphatase of type 2C, is a novel stress signal regulator that inactivates the stress-responsive MAPKs MPK4 and MPK6. Mutant ap2c1 plants produce significantly higher amounts of jasmonate upon wounding and are more resistant to phytophagous mites (Tetranychus urticae). Plants with increased AP2C1 levels display lower wound activation of MAPKs, reduced ethylene production, and compromised innate immunity against the necrotrophic pathogen Botrytis cinerea. Our results demonstrate a key role for the AP2C1 phosphatase in regulating stress hormone levels, defense responses, and MAPK activities in Arabidopsis and provide evidence that the activity of AP2C1 might control the plants response to B. cinerea.

Transgenic Arabidopsis Plants Expressing a Fungal Cutinase Show Alterations in the Structure and Properties of the Cuticle and Postgenital Organ Fusions

A major structural component of the cuticle of plants is cutin. Analysis of the function of cutin in vivo has been limited because no mutants with specific defects in cutin have been characterized. Therefore, transgenic Arabidopsis plants were generated that express and secrete a cutinase from Fusarium solani f sp pisi. Arabidopsis plants expressing the cutinase in the extracellular space showed an altered ultrastructure of the cuticle and an enhanced permeability of the cuticle to solutes. In addition, pollen could germinate on fully differentiated leaves of cutinase-expressing plants but not on control leaves. These differences coincided with strong postgenital organ fusions. The junctions of the fusions contained pectic polysaccharides. As fused organs grew apart from each other, organ deformations and protrusions of epidermal cells developed at positions with high mechanical stress. These results demonstrate that an intact cutin layer not only is important for plant–environment interactions but also prevents fusions between different plant organs and is therefore necessary for normal epidermal differentiation and organ formation.

Nanogram amounts of salicylic acid produced by the rhizobacterium Pseudomonas aeruginosa 7NSK2 activate the systemic acquired resistance pathway in bean.

Root colonization by specific nonpathogenic bacteria can induce a systemic resistance in plants to pathogen infections. In bean, this kind of systemic resistance can be induced by the rhizobacterium Pseudomonas aeruginosa 7NSK2 and depends on the production of salicylic acid by this strain. In a model with plants grown in perlite we demonstrated that Pseudomonas aeruginosa 7NSK2-induced resistance is equivalent to the inclusion of 1 nM salicylic acid in the nutrient solution and used the latter treatment to analyze the molecular basis of this phenomenon. Hydroponic feeding of 1 nM salicylic acid solutions induced phenylalanine ammonia-lyase activity in roots and increased free salicylic acid levels in leaves. Because pathogen-induced systemic acquired resistance involves similar changes it was concluded that 7NSK2-induced resistance is mediated by the systemic acquired resistance pathway. This conclusion was validated by analysis of phenylalanine ammonia-lyase activity in roots and of salicylic acid levels in leaves of soil-grown plants treated with Pseudomonas aeruginosa. The induction of systemic acquired resistance by nanogram amounts of salicylic acid is discussed with respect to long-distance signaling in systemic acquired resistance.

Characterization and biological function of the ISOCHORISMATE SYNTHASE2 gene of Arabidopsis.

Salicylic acid (SA) is an important mediator of plant defense response. In Arabidopsis (Arabidopsis thaliana), this compound was proposed to derive mainly from isochorismate, itself produced from chorismate through the activity of ISOCHORISMATE SYNTHASE1 (ICS1). Null ics1 mutants still accumulate some SA, suggesting the existence of an enzymatic activity redundant with ICS1 or of an alternative ICS-independent SA biosynthetic route. Here, we studied the role of ICS2, a second ICS gene of the Arabidopsis genome, in the production of SA. We have shown that ICS2 encodes a functional ICS enzyme and that, similar to ICS1, ICS2 is targeted to the plastids. Comparison of SA accumulation in the ics1, ics2, and ics1 ics2 mutants indicates that ICS2 participates in the synthesis of SA, but in limited amounts that become clearly detectable only when ICS1 is lacking. This unequal redundancy relationship was also observed for phylloquinone, another isochorismate-derived end product. Furthermore, detection of SA in the double ics1 ics2 double mutant that is completely devoid of phylloquinone provides genetic evidence of the existence of an ICS-independent SA biosynthetic pathway in Arabidopsis.

The glutathione‐deficient mutant pad2‐1 accumulates lower amounts of glucosinolates and is more susceptible to the insect herbivore Spodoptera littoralis

Summary Plants often respond to pathogen or insect attack by inducing the synthesis of toxic compounds such as phytoalexins and glucosinolates (GS). The Arabidopsis mutant pad2-1 has reduced levels of the phytoalexin camalexin and is known for its increased susceptibility to fungal and bacterial pathogens. We found that pad2-1 is also more susceptible to the generalist insect Spodoptera littoralis but not to the specialist Pieris brassicae. The PAD2 gene encodes a gamma-glutamylcysteine synthetase that is involved in glutathione (GSH) synthesis, and consequently the pad2-1 mutant contains about 20% of the GSH found in wild-type plants. Lower GSH levels of pad2-1 were correlated with reduced accumulation of the two major indole and aliphatic GSs of Arabidopsis, indolyl-3-methyl-GS and 4-methylsulfinylbutyl-GS, in response to insect feeding. This effect was specific to GSH, was not complemented by treatment of pad2-1 with the strong reducing agent dithiothreitol, and was not observed with the ascorbate-deficient mutant vtc1-1. In contrast to the jasmonate-insensitive mutant coi1-1, expression of insect-regulated and GS biosynthesis genes was not affected in pad2-1. Our data suggest a crucial role for GSH in GS biosynthesis and insect resistance.

Salicylic acid production in response to biotic and abiotic stress depends on isochorismate in Nicotiana benthamiana

Salicylic acid (SA) is an important signal involved in the activation of defence responses against abiotic and biotic stress. In tobacco, benzoic acid or glucosyl benzoate were proposed to be precursors of SA. This is in sharp contrast with studies in Arabidopsis thaliana, where SA derives from isochorismate. We have determined the importance of isochorismate for SA biosynthesis in Nicotiana benthamiana using virus‐induced gene silencing of the isochorismate synthase (ICS) gene. Plants with silenced ICS expression do not accumulate SA after exposure to UV or to pathogen stress. Plants with silenced ICS expression also exhibit strongly decreased levels of phylloquinone, a product of isochorismate. Our data provide evidence for an isochorismate‐derived synthesis of SA in N. benthamiana