Isolation of Natural Fungal Pathogens from Marchantia polymorpha Reveals Antagonism between Salicylic Acid and Jasmonate during Liverwort-Fungus Interactions.

The evolution of adaptive interactions with beneficial, neutral and detrimental microbes was one of the key features enabling plant terrestrialization. Extensive studies have revealed conserved and unique molecular mechanisms underlying plant-microbe interactions across different plant species; however, most insights gleaned to date have been limited to seed plants. The liverwort Marchantia polymorpha, a descendant of early diverging land plants, is gaining in popularity as an advantageous model system to understand land plant evolution. However, studying evolutionary molecular plant-microbe interactions in this model is hampered by the small number of pathogens known to infect M. polymorpha. Here, we describe four pathogenic fungal strains, Irpex lacteus Marchantia-infectious (MI)1, Phaeophlebiopsis peniophoroides MI2, Bjerkandera adusta MI3 and B. adusta MI4, isolated from diseased M. polymorpha. We demonstrate that salicylic acid (SA) treatment of M. polymorpha promotes infection of the I. lacteus MI1 that is likely to adopt a necrotrophic lifestyle, while this effect is suppressed by co-treatment with the bioactive jasmonate in M. polymorpha, dinor-cis-12-oxo-phytodienoic acid (dn-OPDA), suggesting that antagonistic interactions between SA and oxylipin pathways during plant-fungus interactions are ancient and were established already in liverworts.

Development of Inhibitors of Salicylic Acid Signaling.

Salicylic acid (SA) plays important roles in the induction of systemic acquired resistance (SAR) in plants. Determining the mechanism of SAR will extend our understanding of plant defenses against pathogens. We recently reported that PAMD is an inhibitor of SA signaling, which suppresses the expression of the pathogenesis-related PR genes and is expected to facilitate the understanding of SA signaling. However, PAMD strongly inhibits plant growth. To minimize the side effects of PAMD, we synthesized a number of PAMD derivatives, and identified compound 4 that strongly suppresses the expression of the PR genes with fewer adverse effects on plant growth than PAMD. We further showed that the adverse effects on plant growth were partially caused the stabilization of DELLA, which is also related to the pathogen responses. These results indicate that compound 4 would facilitate our understanding of SA signaling and its cross talk with other plant hormones.

Involvement of abscisic acid and salicylic acid in signal cascade regulating bacterial endophyte-induced volatile oil biosynthesis in plantlets of Atractylodes lancea.

The enormous biological diversity of endophytes, coupled with their potential to enhance the production of bioactive metabolites in plants, has driven research efforts focusing on endophytes. However, limited information is available on the impacts of bacterial endophytes on plant secondary metabolism and signaling pathways involved. This work showed that an endophytic Acinetobacter sp. ALEB16, capable of activating accumulation of plant volatile oils, also induced abscisic acid (ABA) and salicylic acid (SA) production in Atractylodes lancea. Pre-treatment of plantlets with biosynthetic inhibitors of ABA or SA blocked the bacterium-induced volatile production. ABA inhibitors suppressed not only the bacterium-induced volatile accumulation but also the induced ABA and SA generation; nevertheless, SA inhibitors did not significantly inhibit the induced ABA biosynthesis, implying that SA acted downstream of ABA production. These results were confirmed by observations that exogenous ABA and SA reversed the inhibition of bacterium-induced volatile accumulation by inhibitors. Transcriptional activities of genes in sesquiterpenoid biosynthesis also increased significantly with bacterium, ABA and SA treatments. Mevalonate pathway proved to be the main source of isopentenyldiphosphate for bacterium-induced sesquiterpenoids, as assessed in experiments using specific terpene biosynthesis inhibitors. These results suggest that Acinetobacter sp. acts as an endophytic elicitor to stimulate volatile biosynthesis of A. lancea via an ABA/SA-dependent pathway, thereby yielding additional insight into the interconnection between ABA and SA in biosynthesis-related signaling pathways.

Jasmonic acid is involved in the signaling pathway for fungal endophyte-induced volatile oil accumulation of Atractylodes lancea plantlets.

BACKGROUND: Jasmonic acid (JA) is a well-characterized signaling molecule in plant defense responses. However, its relationships with other signal molecules in secondary metabolite production induced by endophytic fungus are largely unknown. Atractylodes lancea (Asteraceae) is a traditional Chinese medicinal plant that produces antimicrobial volatiles oils. We incubated plantlets of A. lancea with the fungus Gilmaniella sp. AL12. to research how JA interacted with other signal molecules in volatile oil production. RESULTS: Fungal inoculation increased JA generation and volatile oil accumulation. To investigate whether JA is required for volatile oil production, plantlets were treated with JA inhibitors ibuprofen (IBU) and nordihydroguaiaretic acid. The inhibitors suppressed both JA and volatile oil production, but fungal inoculation could still induce volatile oils. Plantlets were further treated with the nitric oxide (NO)-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), the H2O2 inhibitors diphenylene iodonium (DPI) and catalase (CAT), and the salicylic acid (SA) biosynthesis inhibitors paclobutrazol and 2-aminoindan-2-phosphonic acid. With fungal inoculation, IBU did not inhibit NO production, and JA generation was significantly suppressed by cPTIO, showing that JA may act as a downstream signal of the NO pathway. Exogenous H2O2 could reverse the inhibitory effects of cPTIO on JA generation, indicating that NO mediates JA induction by the fungus through H2O2-dependent pathways. With fungal inoculation, the H2O2 scavenger DPI/CAT could inhibit JA generation, but IBU could not inhibit H2O2 production, implying that H2O2 directly mediated JA generation. Finally, JA generation was enhanced when SA production was suppressed, and vice versa. CONCLUSIONS: Jasmonic acid acts as a downstream signaling molecule in NO- and H2O2-mediated volatile oil accumulation induced by endophytic fungus and has a complementary interaction with the SA signaling pathway.

Antagonistic plant defense system regulated by phytohormones assists interactions among vector insect, thrips and a tospovirus.

The western flower thrips (Frankliniella occidentalis) is a polyphagous herbivore that causes serious damage to many agricultural plants. In addition to causing feeding damage, it is also a vector insect that transmits tospoviruses such as Tomato spotted wilt virus (TSWV). We previously reported that thrips feeding on plants induces a jasmonate (JA)-regulated plant defense, which negatively affects both the performance and preference (i.e. host plant attractiveness) of the thrips. The antagonistic interaction between a JA-regulated plant defense and a salicylic acid (SA)-regulated plant defense is well known. Here we report that TSWV infection allows thrips to feed heavily and multiply on Arabidopsis plants. TSWV infection elevated SA contents and induced SA-regulated gene expression in the plants. On the other hand, TSWV infection decreased the level of JA-regulated gene expression induced by thrips feeding. Importantly, we also demonstrated that thrips significantly preferred TSWV-infected plants to uninfected plants. In JA-insensitive coi1-1 mutants, however, thrips did not show a preference for TSWV-infected plants. In addition, SA application to wild-type plants increased their attractiveness to thrips. Our results suggest the following mechanism: TSWV infection suppresses the anti-herbivore response in plants and attracts its vector, thrips, to virus-infected plants by exploiting the antagonistic SA-JA plant defense systems.

SA-ABA antagonism in defense responses.

Until recently, phytohormones were mostly studied separately. However, recent studies have suggested that the signaling pathways involved are highly interconnected. We recently reported the antagonistic effects of salicylic acid (SA) and abscisic acid (ABA) in the lesion mimic mutants, cpr22 and ssi4. After shifting these mutants from high humidity, where the lesion mimic phenotype is suppressed to permissive low humidity condition, both SA and ABA pathways were up-regulated. However, the increased levels of SA were able to block downstream ABA responses even though ABA signaling genes and endogenous ABA were elevated. Furthermore, these lesion mimic mutants displayed a partial ABA insensitivity with respect to germination, guard cell opening, and water loss. This increased water loss in detached mutant plants could also be mimicked by treating wild type plants with SA. An active SA analog, 5-chloro-salicylic acid also induced enhanced water loss, while an inactive analog, 4-hydroxy-benzoic acid, did not. Here, we report that the biological analogs of SA, the systemic acquired resistance (SAR) activators, BTH (benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester) and BIT (1,2-benzisothiazol-3(2H)-one1,1-dioxide), did not have the same effect as SA, suggesting that SA may have additional roles to defense, and that SAR activators may not mimic all SA effects.

Novel interrelationship between salicylic acid, abscisic acid, and PIP2-specific phospholipase C in heat acclimation-induced thermotolerance in pea leaves.

Increasing evidence suggests that heat acclimation and exogenous salicylic acid (SA) and abscisic acid (ABA) may lead to the enhancement of thermotolerance in plants. In this study, the roles that free SA, conjugated SA, ABA, and phosphatidylinositol-4,5-bisphosphate (PIP(2))-specific phospholipase C (PLC) play in thermotolerance development induced by heat acclimation (38 degrees C) were investigated. To evaluate their potential functions, three inhibitors of synthesis or activity were infiltrated into pea leaves prior to heat acclimation treatment. The results showed that the burst of free SA in response to heat acclimation could be attributed to the conversion of SA 2-O-D-glucose, the main conjugated form of SA, to free SA. Inhibition of ABA biosynthesis also resulted in a defect in the free SA peak during heat acclimation. In acquired thermotolerance assessment, the greatest weakness of antioxidant enzyme activity and the most severe heat injury (malondialdehyde content and degree of wilting) were found in pea leaves pre-treated with neomycin, a well-known inhibitor of PIP(2)-PLC activity. PsPLC gene expression was activated by exogenous ABA, SA treatments, and heat acclimation after pre-treatments with a SA biosynthesis inhibitor. From these results, PIP(2)-PLC appears to play a key role in free SA- and ABA-associated reinforcement of thermotolerance resulting from heat acclimation.

The outcomes of concentration-specific interactions between salicylate and jasmonate signaling include synergy, antagonism, and oxidative stress leading to cell death.

Salicylic acid (SA) has been proposed to antagonize jasmonic acid (JA) biosynthesis and signaling. We report, however, that in salicylate hydroxylase-expressing tobacco (Nicotiana tabacum) plants, where SA levels were reduced, JA levels were not elevated during a hypersensitive response elicited by Pseudomonas syringae pv phaseolicola. The effects of cotreatment with various concentrations of SA and JA were assessed in tobacco and Arabidopsis (Arabidopsis thaliana). These suggested that there was a transient synergistic enhancement in the expression of genes associated with either JA (PDF1.2 [defensin] and Thi1.2 [thionin]) or SA (PR1 [PR1a-beta-glucuronidase in tobacco]) signaling when both signals were applied at low (typically 10-100 microm) concentrations. Antagonism was observed at more prolonged treatment times or at higher concentrations. Similar results were also observed when adding the JA precursor, alpha-linolenic acid with SA. Synergic effects on gene expression and plant stress were NPR1- and COI1-dependent, SA- and JA-signaling components, respectively. Electrolyte leakage and Evans blue staining indicated that application of higher concentrations of SA + JA induced plant stress or death and elicited the generation of apoplastic reactive oxygen species. This was indicated by enhancement of hydrogen peroxide-responsive AoPR10-beta-glucuronidase expression, suppression of plant stress/death using catalase, and direct hydrogen peroxide measurements. Our data suggests that the outcomes of JA-SA interactions could be tailored to pathogen/pest attack by the relative concentration of each hormone.

A family of conserved bacterial effectors inhibits salicylic acid-mediated basal immunity and promotes disease necrosis in plants.

Salicylic acid (SA)-mediated host immunity plays a central role in combating microbial pathogens in plants. Inactivation of SA-mediated immunity, therefore, would be a critical step in the evolution of a successful plant pathogen. It is known that mutations in conserved effector loci (CEL) in the plant pathogens Pseudomonas syringae (the Delta CEL mutation), Erwinia amylovora (the dspA/E mutation), and Pantoea stewartii subsp. stewartii (the wtsE mutation) exert particularly strong negative effects on bacterial virulence in their host plants by unknown mechanisms. We found that the loss of virulence in Delta CEL and dspA/E mutants was linked to their inability to suppress cell wall-based defenses and to cause normal disease necrosis in Arabidopsis and apple host plants. The Delta CEL mutant activated SA-dependent callose deposition in wild-type Arabidopsis but failed to elicit high levels of callose-associated defense in Arabidopsis plants blocked in SA accumulation or synthesis. This mutant also multiplied more aggressively in SA-deficient plants than in wild-type plants. The hopPtoM and avrE genes in the CEL of P. syringae were found to encode suppressors of this SA-dependent basal defense. The widespread conservation of the HopPtoM and AvrE families of effectors in various bacteria suggests that suppression of SA-dependent basal immunity and promotion of host cell death are important virulence strategies for bacterial infection of plants.

Salicylic acid-independent induction of pathogenesis-related gene expression by fusicoccin.

Treatment of tomato plants (Lycopersicon esculentum Mill.) with fusicoccin (FC), an activator of the plasma-membrane H+-ATPase which maintains an electrochemical gradient across the plasma membrane, resulted in a dose-dependent accumulation of transcripts for intra- and extracellular pathogenesis-related (PR) proteins. The accumulation of PR protein transcripts was paralleled by an increase in leaf salicylic acid (SA) content. Transcripts of PR proteins and SA started to accumulate 3 h after FC treatment. 2-Aminoindan-2-phosphonic acid, an inhibitor of SA synthesis, was used to assess the role of SA in FC-mediated induction of PR gene expression. 2-Aminoindan-2-phosphonic acid was found to suppress the accumulation of SA but not the induction of PR gene expression in response to FC treatment. Furthermore, in transgenic tobacco plants overexpressing a bacterial salicylate hydroxylase gene (nahG-tobacco), PR transcripts accumulated after FC treatment to levels similar to those observed in control tobacco plants. The data indicate a role for the proton gradient across the plasma membrane in the SA-independent induction of PR gene expression.

Salicylate-induced kidney mitochondrial permeability transition is prevented by cyclosporin A.

The effect of salicylate, the active metabolite of aspirin (acetyl salicylic acid) in the presence of Ca2+ and phosphate on mitochondrial permeability transition (MPT) was studied. MPT is often associated with opening of a Ca2+ -induced pore. The opening of this pore leads to swelling, loss of mitochondrial membrane potential and release of accumulated Ca2+. In freshly isolated rat kidney mitochondria, salicylate (400 microM) in the presence of 20 nmol Ca2+/mg protein and 0.1 mM phosphate induced swelling, loss of mitochondrial membrane potential and release of accumulated Ca2+. All these changes were eliminated when cyclosporin A (1 microM), (a pore inhibitory agent) was included in the incubation medium. Unlike salicylate, unhydrolyzed aspirin (400 microM) induced these changes slightly. We concluded that salicylate acts as an activator of Ca2+ and phosphate in promoting the opening of kidney inner mitochondrial membrane pore. As a result a great consideration should be given to its toxicological effect.