beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophic fungus Botrytis cinerea

The non-protein amino acid beta-aminobutyric acid (BABA) protects numerous plants against various pathogens. Protection of Arabidopsis plants against virulent pathogens involves the potentiation of pathogen-specific defense responses. To extend the analysis of the mode of action of BABA to necrotrophs we evaluated the effect of this chemical on Arabidopsis plants infected with the gray mold fungus Botrytis cinerea. BABA-treated Arabidopsis were found to be less sensitive to two different strains of this pathogen. BABA protected mutants defective in the jasmonate and ethylene pathways, but was inactive in plants impaired in the systemic acquired resistance transduction pathway. Treatments with benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester, a functional analog of salicylic acid (SA), also markedly reduced the level of infection. Moreover, BABA potentiated mRNA accumulation of the SA-associated PR-1, but not the jasmonate/ethylene-dependent PDF1.2 gene. Thus, besides jasmonate/ethylene-dependent defense responses, SA-dependent signaling also contributes to restrict B. cinerea infection in Arabidopsis. Our results also suggest that SA-dependent signaling is down-regulated after infection by B. cinerea. The observed up-regulation of the PDF1.2 gene in mutants defective in the SA-dependent signaling pathway points to a cross-talk between SA- and jasmonate/ethylene-dependent signaling pathways during pathogen ingress.     

Nuclear genes that promote splicing of group I introns in the chloroplast 23S rRNA and psbA genes in Chlamydomonas reinhardtii

Defence against pathogens in Arabidopsis is orchestrated by at least three signalling molecules: salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). The hrl1 (hypersensitive response-like lesions 1) mutant of Arabidopsis is characterized by spontaneous necrotic lesions, accumulation of reactive oxygen species, constitutive expression of SA- and ET/JA-responsive defence genes, and enhanced resistance to virulent bacterial and oomycete pathogens. Epistasis analyses of hrl1 with npr1, etr1, coi1 and SA-depleted nahG plants revealed novel interactions between SA and ET/JA signalling pathways in regulating defence gene expression and cell death. RNA gel-blot analysis of RNA isolated separately from the lesion+ and the lesion- leaves of double mutants of hrl1 revealed different signalling requirements for the expression of defence genes in these tissues. Expression of the ET/JA-responsive PDF1.2 gene was markedly reduced in hrl1 npr1 and in SA-depleted hrl1 nahG plants. In hrl1 nahG plants, expression of PDF1.2 was regulated by benzathiadiazole in a concentration-dependent manner: induced at low concentration and suppressed at high concentration. The hrl1 etr1 plants lacked systemic PR-1 expression, and exhibited compromised resistance to virulent Pseudomonas syringae and Peronospora parasitica. Inhibiting JA responses in hrl1 coi1 plants lead to exaggerated cell death and severe stunting of plants. Finally, the hrl1 mutation lead to elevated expression of AtrbohD, which encodes a major subunit of the NADPH oxidase complex. Our results indicate that defence gene expression and resistance against pathogens in hrl1 is regulated synergistically by SA and ET/JA defence pathways.     

Susceptible to intolerance--a range of hormonal actions in a susceptible Arabidopsis pathogen response

Ethylene and salicylic acid (SA) are key intermediates in a host's response to pathogens. Previously, we have shown using a tomato compatible interaction that ethylene and SA act sequentially and are essential for disease symptom production. Here, we have examined the relationship between the two signals in the Arabidopsis-Xanthomonas campestris pv. campestris (Xcc) compatible interaction. Preventing SA accumulation by expression of the nahG gene reduced subsequent ethylene production and altered the development of disease symptoms, with plants showing no visible chlorosis. The ethylene insensitive lines, etr1-1 and etr2-1, on the other hand, accumulated SA and exhibited normal but precocious symptom development. Therefore, Arabidopsis, like tomato, was found to exhibit co-operative ethylene and SA action for the production of disease symptoms. However, in Arabidopsis, SA was found to act upstream of ethylene. Jasmonic acid and indole-3-acetic acid levels were also found to increase in response to Xcc. In contrast to ethylene, accumulation of these hormones was not found to be dependent on SA action. These results indicate that the plants response to a virulent pathogen is a composite of multiple signaling pathways.     

Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway.

A 5-kD plant defensin was purified from Arabidopsis leaves challenged with the fungus Alternaria brassicicola and shown to possess antifungal properties in vitro. The corresponding plant defensin gene was induced after treatment of leaves with methyl jasmonate or ethylene but not with salicylic acid or 2,6-dichloroisonicotinic acid. When challenged with A. brassicicola, the levels of the plant defensin protein and mRNA rose both in inoculated leaves and in nontreated leaves of inoculated plants (systemic leaves). These events coincided with an increase in the endogenous jasmonic acid content of both types of leaves. Systemic pathogen-induced expression of the plant defensin gene was unaffected in Arabidopsis transformants (nahG) or mutants (npr1 and cpr1) affected in the salicylic acid response but was strongly reduced in the Arabidopsis mutants eln2 and col1 that are blocked in their response to ethylene and methyl jasmonate, respectively. Our results indicate that systemic pathogen-induced expression of the plant defensin gene in Arabidopsis is independent of salicylic acid but requires components of the ethylene and jasmonic acid response.     

The plant growth-promoting rhizobacterium Bacillus cereus AR156 induces systemic resistance in Arabidopsis thaliana by simultaneously activating salicylate- and jasmonate/ethylene-dependent signaling pathways

Bacillus cereus AR156 is a plant growth-promoting rhizobacterium that induces resistance against a broad spectrum of pathogens including Pseudomonas syringae pv. tomato DC3000. This study analyzed AR156-induced systemic resistance (ISR) to DC3000 in Arabidopsis ecotype Col-0 plants. Compared with mock-treated plants, AR156-treated ones showed an increase in biomass and reductions in disease severity and pathogen density in the leaves. The defense-related genes PR1, PR2, PR5, and PDF1.2 were concurrently expressed in the leaves of AR156-treated plants, suggesting simultaneous activation of the salicylic acid (SA)- and the jasmonic acid (JA)- and ethylene (ET)-dependent signaling pathways by AR156. The above gene expression was faster and stronger in plants treated with AR156 and inoculated with DC3000 than that in plants only inoculated with DC3000. Moreover, the cellular defense responses hydrogen peroxide accumulation and callose deposition were induced upon challenge inoculation in the leaves of Col-0 plants primed by AR156. Also, pretreatment with AR156 led to a higher level of induced protection against DC3000 in Col-0 than that in the transgenic NahG, the mutant jar1 or etr1, but the protection was absent in the mutant npr1. Therefore, AR156 triggers ISR in Arabidopsis by simultaneously activating the SA- and JA/ET-signaling pathways in an NPR1-dependent manner that leads to an additive effect on the level of induced protection.     

Enhancing Arabidopsis salt and drought stress tolerance by chemical priming for its abscisic acid responses

Drought and salt stress tolerance of Arabidopsis (Arabidopsis thaliana) plants increased following treatment with the nonprotein amino acid beta-aminobutyric acid (BABA), known as an inducer of resistance against infection of plants by numerous pathogens. BABA-pretreated plants showed earlier and higher expression of the salicylic acid-dependent PR-1 and PR-5 and the abscisic acid (ABA)-dependent RAB-18 and RD-29A genes following salt and drought stress. However, non-expressor of pathogenesis-related genes 1 and constitutive expressor of pathogenesis-related genes 1 mutants as well as transgenic NahG plants, all affected in the salicylic acid signal transduction pathway, still showed increased salt and drought tolerance after BABA treatment. On the contrary, the ABA deficient 1 and ABA insensitive 4 mutants, both impaired in the ABA-signaling pathway, could not be protected by BABA application. Our data demonstrate that BABA-induced water stress tolerance is based on enhanced ABA accumulation resulting in accelerated stress gene expression and stomatal closure. Here, we show a possibility to increase plant tolerance for these abiotic stresses through effective priming of the preexisting defense pathways without resorting to genetic alterations.     

The Arabidopsis flavin-dependent monooxygenase FMO1 is an essential component of biologically induced systemic acquired resistance

Upon localized attack by necrotizing pathogens, plants gradually develop increased resistance against subsequent infections at the whole-plant level, a phenomenon known as systemic acquired resistance (SAR). To identify genes involved in the establishment of SAR, we pursued a strategy that combined gene expression information from microarray data with pathological characterization of selected Arabidopsis (Arabidopsis thaliana) T-DNA insertion lines. A gene that is up-regulated in Arabidopsis leaves inoculated with avirulent or virulent strains of the bacterial pathogen Pseudomonas syringae pv maculicola (Psm) showed homology to flavin-dependent monooxygenases (FMO) and was designated as FMO1. An Arabidopsis knockout line of FMO1 proved to be fully impaired in the establishment of SAR triggered by avirulent (Psm avrRpm1) or virulent (Psm) bacteria. Loss of SAR in the fmo1 mutants was accompanied by the inability to initiate systemic accumulation of salicylic acid (SA) and systemic expression of diverse defense-related genes. In contrast, responses at the site of pathogen attack, including increases in the levels of the defense signals SA and jasmonic acid, camalexin accumulation, and expression of various defense genes, were induced in a similar manner in both fmo1 mutant and wild-type plants. Consistently, the fmo1 mutation did not significantly affect local disease resistance toward virulent or avirulent bacteria in naive plants. Induction of FMO1 expression at the site of pathogen inoculation is independent of SA signaling, but attenuated in the Arabidopsis eds1 and pad4 defense mutants. Importantly, FMO1 expression is also systemically induced upon localized P. syringae infection. This systemic up-regulation is missing in the SAR-defective SA pathway mutants sid2 and npr1, as well as in the defense mutant ndr1, indicating a close correlation between systemic FMO1 expression and SAR establishment. Our findings suggest that the presence of the FMO1 gene product in systemic tissue is critical for the development of SAR, possibly by synthesis of a metabolite required for the transduction or amplification of a signal during the early phases of SAR establishment in systemic leaves.     

Probenazole induces systemic acquired resistance in Arabidopsis with a novel type of action

Probenazole (PBZ; 3-allyloxy-1,2-benzisothiazole-1,1-dioxide), which is the active ingredient in Oryzemate, has been used widely in Asia to protect rice plants against the rice blast fungus Magnaporthe grisea. To study PBZ's mode of action, we analyzed its ability, as well as that of its active metabolite 1, 2-benzisothiazol-3 (2H)-one 1,1-dioxide (BIT) to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild-type Arabidopsis treated with PBZ or BIT exhibited increased expression of several pathogenesis-related genes, increased levels of total salicylic acid (SA), and enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC 3000 and the oomycete pathogen Peronospora parasitica Emco5. The role of several defense signaling hormones, such as SA, ethylene and jasmonic acid (JA), in activating resistance following PBZ or BIT treatment was analyzed using NahG transgenic plants and etr1-1 and coi1-1 mutant plants, respectively. In addition, the involvement of NPR1, a key component in the SA signaling pathway leading to defense responses, was assessed. PBZ or BIT treatment did not induce disease resistance or PR-1 expression in NahG transgenic or npr1 mutant plants, but it did activate these phenomena in etr1-1 and coi 1-1 mutant plants. Thus SA and NPR1 appear to be required for PBZ- and BIT-mediated activation of defense responses, while ethylene and JA are not. Furthermore, our data suggest that PBZ and BIT comprise a novel class of defense activators that stimulate the SA/NPR1-mediated defense signaling pathway upstream of SA.     

The Arabidopsis ssi1 mutation restores pathogenesis-related gene expression in npr1 plants and renders defensin gene expression salicylic acid dependent.

The Arabidopsis NPR1 gene was previously shown to be required for the salicylic acid (SA)- and benzothiadiazole (BTH)-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance. The dominant ssi1 (for suppressor of SA insensitivity) mutation characterized in this study defines a new component of the SA signal transduction pathway that bypasses the requirement of NPR1 for expression of the PR genes and disease resistance. The ssi1 mutation caused PR (PR-1, BGL2 [PR-2], and PR-5) genes to be constitutively expressed and restored resistance to an avirulent strain of Pseudomonas syringae pv tomato in npr1-5 (previously called sai1) mutant plants. In addition, ssi1 plants were small, spontaneously developed hypersensitive response-like lesions, accumulated elevated levels of SA, and constitutively expressed the antimicrobial defensin gene PDF1.2. The phenotypes of the ssi1 mutant are SA dependent. When SA accumulation was prevented in ssi1 npr1-5 plants by expressing the SA-degrading salicylate hydroxylase (nahG) gene, all of the phenotypes associated with the ssi1 mutation were suppressed. However, lesion formation and expression of the PR genes were restored in these plants by the application of BTH. Interestingly, expression of PDF1.2, which previously has been shown to be SA independent but jasmonic acid and ethylene dependent, was also suppressed in ssi1 npr1-5 plants by the nahG gene. Furthermore, exogenous application of BTH restored PDF1.2 expression in these plants. Our results suggest that SSI1 may function as a switch modulating cross-talk between the SA- and jasmonic acid/ethylene-mediated defense signal transduction pathways.     

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.     

Topology of the network integrating salicylate and jasmonate signal transduction derived from global expression phenotyping

The signal transduction network controlling plant responses to pathogens includes pathways requiring the signal molecules salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). The network topology was explored using global expression phenotyping of wild-type and signaling-defective mutant plants, including eds3, eds4, eds5, eds8, pad1, pad2, pad4, NahG, npr1, sid2, ein2, and coi1. Hierarchical clustering was used to define groups of mutations with similar effects on gene expression and groups of similarly regulated genes. Mutations affecting SA signaling formed two groups: one comprised of eds4, eds5, sid2, and npr1-3 affecting only SA signaling; and the other comprised of pad2, eds3, npr1-1, pad4, and NahG affecting SA signaling as well as another unknown process. Major differences between the expression patterns in NahG and the SA biosynthetic mutant sid2 suggest that NahG has pleiotropic effects beyond elimination of SA. A third group of mutants comprised of eds8, pad1, ein2, and coi1 affected ethylene and jasmonate signaling. Expression patterns of some genes revealed mutual inhibition between SA- and JA-dependent signaling, while other genes required JA and ET signaling as well as the unknown signaling process for full expression. Global expression phenotype similarities among mutants suggested, and experiments confirmed, that EDS3 affects SA signaling while EDS8 and PAD1 affect JA signaling. This work allowed modeling of network topology, definition of co-regulated genes, and placement of previously uncharacterized regulatory genes in the network.     

Acquired Resistance Signal Transduction in Arabidopsis Is Ethylene Independent

To clarify the role of ethylene in systemic acquired resistance (SAR), we conducted experiments using Arabidopsis ethylene response mutants. Plants that are nonresponsive to ethylene (i.e., [theta]tr1 and [theta]in2) showed normal sensitivity to the SAR-inducing chemicals salicylic acid (SA) and 2,6-dichloroisonicotinic acid with respect to SAR gene induction and pathogen resistance. This indicated that chemically induced SAR is not an ethylene-dependent process in Arabidopsis. Ethephon, an ethylene-releasing chemical, induced SAR gene expression in both the wild type and ethylene mutants, whereas ethylene alone did not, suggesting that induction of these genes by ethephon is not due to the action of ethylene. Furthermore, transgenic plants expressing salicylate hydroxylase, a bacterial enzyme that degrades SA to catechol, did not accumulate SAR mRNAs in response to ethephon. Thus, SAR gene induction by ethephon appears to be mediated through SA. Other experiments suggested that ethylene may play a role in SAR by enhancing tissue sensitivity to the action of SA.