A critical role for Arabidopsis MILDEW RESISTANCE LOCUS O2 in systemic acquired resistance

Members of the MILDEW RESISTANCE LOCUS O (MLO) gene family confer susceptibility to powdery mildews in different plant species, and their existence therefore seems to be disadvantageous for the plant. We recognized that expression of the Arabidopsis MLO2 gene is induced after inoculation with the bacterial pathogen Pseudomonas syringae, promoted by salicylic acid (SA) signaling, and systemically enhanced in the foliage of plants exhibiting systemic acquired resistance (SAR). Importantly, distinct mlo2 mutant lines were unable to systemically increase resistance to bacterial infection after inoculation with P. syringae, indicating that the function of MLO2 is necessary for biologically induced SAR in Arabidopsis. Our data also suggest that the close homolog MLO6 has a supportive but less critical role in SAR. In contrast to SAR, basal resistance to bacterial infection was not affected in mlo2. Remarkably, SAR‐defective mlo2 mutants were still competent in systemically increasing the levels of the SAR‐activating metabolites pipecolic acid (Pip) and SA after inoculation, and to enhance SAR‐related gene expression in distal plant parts. Furthermore, although MLO2 was not required for SA‐ or Pip‐inducible defense gene expression, it was essential for the proper induction of disease resistance by both SAR signals. We conclude that MLO2 acts as a critical downstream component in the execution of SAR to bacterial infection, being required for the translation of elevated defense responses into disease resistance. Moreover, our data suggest a function for MLO2 in the activation of plant defense priming during challenge by P. syringae.     

Induced systemic resistance (ISR) in plants: mechanism of action

Plants possess a range of active defense apparatuses that can be actively expressed in response to biotic stresses (pathogens and parasites) of various scales (ranging from microscopic viruses to phytophagous insect). The timing of this defense response is critical and reflects on the difference between coping and succumbing to such biotic challenge of necrotizing pathogens/parasites. If defense mechanisms are triggered by a stimulus prior to infection by a plant pathogen, disease can be reduced. Induced resistance is a state of enhanced defensive capacity developed by a plant when appropriately stimulated. Systemic acquired resistance (SAR) and induced systemic resistance (ISR) are two forms of induced resistance wherein plant defenses are preconditioned by prior infection or treatment that results in resistance against subsequent challenge by a pathogen or parasite. Selected strains of plant growth-promoting rhizobacteria (PGPR) suppress diseases by antagonism between the bacteria and soil-borne pathogens as well as by inducing a systemic resistance in plant against both root and foliar pathogens. Rhizobacteria mediated ISR resembles that of pathogen induced SAR in that both types of induced resistance render uninfected plant parts more resistant towards a broad spectrum of plant pathogens. Several rhizobacteria trigger the salicylic acid (SA)-dependent SAR pathway by producing SA at the root surface whereas other rhizobacteria trigger different signaling pathway independent of SA. The existence of SA-independent ISR pathway has been studied in Arabidopsis thaliana, which is dependent on jasmonic acid (JA) and ethylene signaling. Specific Pseudomonas strains induce systemic resistance in viz., carnation, cucumber, radish, tobacco, and Arabidopsis, as evidenced by an enhanced defensive capacity upon challenge inoculation. Combination of ISR and SAR can increase protection against pathogens that are resisted through both pathways besides extended protection to a broader spectrum of pathogens than ISR/SAR alone. Beside Pseudomonas strains, ISR is conducted by Bacillus spp. wherein published results show that several specific strains of species B. amyloliquifaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B.sphaericus elicit significant reduction in the incidence or severity of various diseases on a diversity of hosts.     

The Elongator complex‐associated protein DRL1 plays a positive role in immune responses against necrotrophic fungal pathogens in Arabidopsis

DEFORMED ROOT AND LEAVES1 (DRL1) is an Arabidopsis homologue of the yeast TOXIN TARGET4 (TOT4)/KILLER TOXIN‐INSENSITIVE12 (KTI12) protein that is physically associated with the RNA polymerase II‐interacting protein complex named Elongator. Mutations in DRL1 and Elongator lead to similar morphological and molecular phenotypes, suggesting that DRL1 and Elongator may functionally overlap in Arabidopsis. We have shown previously that Elongator plays an important role in both salicylic acid (SA)‐ and jasmonic acid (JA)/ethylene (ET)‐mediated defence responses. Here, we tested whether DRL1 also plays a similar role as Elongator in plant immune responses. Our results show that, although DRL1 partially contributes to SA‐induced cytotoxicity, it does not play a significant role in SA‐mediated expression of PATHOGENESIS‐RELATED genes and resistance to the virulent bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. In contrast, DRL1 is required for JA/ET‐ and necrotrophic fungal pathogen Botrytis cinerea‐induced defence gene expression and for resistance to B. cinerea and Alternaria brassicicola. Furthermore, unlike the TOT4/KTI12 gene which, when overexpressed in yeast, confers zymocin resistance, a phenotype of the tot4/kti12 mutant, overexpression of DRL1 does not change B. cinerea‐induced defence gene expression and resistance to this pathogen. Finally, DRL1 contains an N‐terminal P‐loop and a C‐terminal calmodulin (CaM)‐binding domain and is a CaM‐binding protein. We demonstrate that both the P‐loop and the CaM‐binding domain are essential for the function of DRL1 in B. cinerea‐induced expression of PDF1.2 and ORA59, and in resistance to B. cinerea, suggesting that the function of DRL1 in plant immunity may be regulated by ATP/GTP and CaM binding.     

Overexpression of a Monocot Acyl‐CoA‐Binding Protein Confers Broad‐Spectrum Pathogen Protection in a Dicot

Plants are continuously infected by various pathogens throughout their lifecycle. Previous studies have reported that the expression of Class III acyl‐CoA‐binding proteins (ACBPs) such as the Arabidopsis ACBP3 and rice ACBP5 were induced by pathogen infection. Transgenic Arabidopsis AtACBP3‐overexpressors (AtACBP3‐OEs) displayed enhanced protection against the bacterial biotroph, Pseudomonas syringae, although they became susceptible to the fungal necrotroph Botrytis cinerea. A Class III ACBP from a monocot, rice (Oryza sativa) OsACBP5 was overexpressed in the dicot Arabidopsis. The resultant transgenic Arabidopsis lines conferred resistance not only to the bacterial biotroph P. syringae but to fungal necrotrophs (Rhizoctonia solani, B. cinerea, Alternaria brassicicola) and a hemibiotroph (Colletotrichum siamense). Changes in protein expression in R. solani‐infected Arabidopsis OsACBP5‐overexpressors (OsACBP5‐OEs) were demonstrated using proteomic analysis. Biotic stress‐related proteins including cell wall‐related proteins such as FASCILIN‐LIKE ARABINOGALACTAN‐PROTEIN10, LEUCINE‐RICH REPEAT EXTENSIN‐LIKE PROTEINS, XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE PROTEIN4, and PECTINESTERASE INHIBITOR18; proteins associated with glucosinolate degradation including GDSL‐LIKE LIPASE23, EPITHIOSPECIFIER MODIFIER1, MYROSINASE1, MYROSINASE2, and NITRILASE1; as well as a protein involved in jasmonate biosynthesis, ALLENE OXIDE CYCLASE2, were induced in OsACBP5‐OEs upon R. solani infection. These results indicated that upregulation of these proteins in OsACBP5‐OEs conferred protection against various plant pathogens.     

Effects of colonization of a bacterial endophyte,Azospirillum sp. B510, on disease resistance in tomato

A plant growth-promoting bacteria, Azospirillum sp. B510, isolated from rice, can enhance growth and yield and induce disease resistance against various types of diseases in rice. Because little is known about the interaction between other plant species and this strain, we have investigated the effect of its colonization on disease resistance in tomato plants. Treatment with this strain by soil-drenching method established endophytic colonization in root tissues in tomato plant. The endophytic colonization with this strain-induced disease resistance in tomato plant against bacterial leaf spot caused by Pseudomonas syringae pv. tomato and gray mold caused by Botrytis cinerea. In Azospirillum-treated plants, neither the accumulation of SA nor the expression of defense-related genes was observed. These indicate that endophytic colonization with Azospirillum sp. B510 is able to activate the innate immune system also in tomato, which does not seem to be systemic acquired resistance.     

CASAR82A, a Pathogen-induced Pepper SAR8.2, Exhibits an Antifungal Activity and its Overexpression Enhances Disease Resistance and Stress Tolerance

Pepper SAR8.2 gene (CASAR82A) was previously reported to be locally or systemically induced in pepper plants by biotic and abiotic stresses. In this study, the physiological and molecular functions of the pepper SAR8.2 protein in the plant defense responses were investigated by generating Arabidopsis transgenic lines overexpressing the CASAR82A gene. The transgenic Arabidopsis plants grew faster than the wild-type plants, indicating that the CASAR82A gene was involved in plant development. The ectopic expression of CASAR82A in Arabidopsis was accompanied by the expression of the Arabidopsis pathogenesis-related (PR)-genes including AtPR-1, AtPR-4 and AtPR-5. CASAR82A overexpression enhanced the resistance against infections by Pseudomonas syringae pv. tomato, Fusarium oxysporum f.sp. matthiolae or Botrytis cinerea. The transgenic plants also exhibited increased NaCl and drought tolerance during all growth stages. Moreover, the methyl viologen test showed that the transgenic plants were tolerant to oxidative stress. The purified recombinant CASAR82A protein and crude protein extracts of the transgenic plants exhibited antifungal activity against some phytopathogenic fungi, indicating that the enhanced resistance of the transgenic plants to fungal pathogen infection may be due to the antifungal effect of SAR8.2 protein.     

Methyl Salicylate Production and Jasmonate Signaling Are Not Essential for Systemic Acquired Resistance in Arabidopsis

Systemic acquired resistance (SAR) develops in response to local microbial leaf inoculation and renders the whole plant more resistant to subsequent pathogen infection. Accumulation of salicylic acid (SA) in noninfected plant parts is required for SAR, and methyl salicylate (MeSA) and jasmonate (JA) are proposed to have critical roles during SAR long-distance signaling from inoculated to distant leaves. Here, we address the significance of MeSA and JA during SAR development in Arabidopsis thaliana. MeSA production increases in leaves inoculated with the SAR-inducing bacterial pathogen Pseudomonas syringae; however, most MeSA is emitted into the atmosphere, and only small amounts are retained. We show that in several Arabidopsis defense mutants, the abilities to produce MeSA and to establish SAR do not coincide. T-DNA insertion lines defective in expression of a pathogen-responsive SA methyltransferase gene are completely devoid of induced MeSA production but increase systemic SA levels and develop SAR upon local P. syringae inoculation. Therefore, MeSA is dispensable for SAR in Arabidopsis, and SA accumulation in distant leaves appears to occur by de novo synthesis via isochorismate synthase. We show that MeSA production induced by P. syringae depends on the JA pathway but that JA biosynthesis or downstream signaling is not required for SAR. In compatible interactions, MeSA production depends on the P. syringae virulence factor coronatine, suggesting that the phytopathogen uses coronatine-mediated volatilization of MeSA from leaves to attenuate the SA-based defense pathway.     

The Botrytis cinerea cerato‐platanin BcSpl1 is a potent inducer of systemic acquired resistance (SAR) in tobacco and generates a wave of salicylic acid expanding from the site of application

Systemic acquired resistance (SAR) is a potent plant defence system that, in response to a first contact with a plant pathogen, prepares the whole plant for subsequent attacks, so that it becomes more resistant to the same and to other pathogens. BcSpl1, a cerato‐platanin family protein abundantly secreted by Botrytis cinerea, is required for full virulence and elicits the hypersensitive response in the host. Here, we report that BcSpl1 is also able to induce in tobacco systemic resistance to two plant pathogens, Pseudomonas syringae and B. cinerea, which correlates with the induction of two pathogenesis‐related genes, PR‐1a and PR‐5. Levels of salicylic acid were quantified in situ on BcSpl1 infiltration, and a wave of salicylic acid departing from the point of infiltration and running through the leaf was observed, as well as the appearance of this plant hormone in the neighbouring leaves as early as 3 days after infiltration.     

Priming for JA-dependent defenses using hexanoic acid is an effective mechanism to protect Arabidopsis against B. cinerea

Soil drench treatments with hexanoic acid can effectively protect Arabidopsis plants against Botrytis cinerea through a mechanism based on a stronger and faster accumulation of JA-dependent defenses.Plants impaired in ethylene, salicylic acid, abscisic acid or glutathion pathways showed intact protection by hexanoic acid upon B. cinerea infection. Accordingly, no significant changes in the SA marker gene PR-1 in either the SA or ABA hormone balance were observed in the infected and treated plants. In contrast, the JA signaling pathway showed dramatic changes after hexanoic acid treatment, mainly when the pathogen was present. The impaired JA mutants, jin1-2 and jar1, were unable to display hexanoic acid priming against the necrotroph. In addition, hexanoic acid-treated plants infected with B. cinerea showed priming in the expression of the PDF1.2, PR-4 and VSP1 genes implicated in the JA pathways. Moreover, JA and OPDA levels were primed at early stages by hexanoic acid. Treatments also stimulated increased callose accumulation in response to the pathogen. Although callose accumulation has proved an effective IR mechanism against B. cinerea, it is apparently not essential to express hexanoic acid-induced resistance (HxAc-IR) because the mutant pmr4.1 (callose synthesis defective mutant) is protected by treatment.We recently described how hexanoic acid treatments can protect tomato plants against B. cinerea by stimulating ABA-dependent callose deposition and by priming OPDA and JA-Ile production. We clearly demonstrate here that Hx-IR is a dependent plant species, since this acid protects Arabidopsis plants against the same necrotroph by priming JA-dependent defenses without enhancing callose accumulation.     

Biologically induced systemic acquired resistance in Arabidopsis thaliana

Local infection with a necrotizing pathogen can render plants resistant to subsequent infection by normally virulent pathogens. A system for biological induction of such systemic acquired resistance (SAR) in Arabidopsis thaliana is reported. When plants were immunized by local inoculation of a single leaf with avirulent Pseudomonas syringae pv. tomato (Pst) carrying the avrRpt2 avirulence gene, after 2 days other leaves became resistant, as measured symptomatically and by in planta bacterial growth, to challenge with a virulent Pst strain lacking this avirulence gene. Resistance was systemic and protected the plants against infection by other virulent pathogens including P. syringae pv. maculicola. Low-dose inoculation induced a strong SAR and double immunizations did not increase the level of protection indicating that the response of only a few cells to the immunizing bacteria is required. SAR was not induced by the virulent strain of Pst lacking avrRpt2. However, experiments with the Arabidopsis RPS2 disease resistance gene mutant rps2-201, which does not exhibit a local hypersensitive response to Pst carrying the corresponding avirulence gene avrRpt2, indicate that a hypersensitive response contributes to, but is not essential for, the induction of SAR. Thus, avrRpt2 activates either a branching signal pathway or separate parallel pathways for induction of localized hypersensitive resistance and SAR, with downstream potentiation of the systemic response by the local response. Using this system for the biological induction of SAR in Arabidopsis, it should be possible to dissect the molecular genetics of SAR by the isolation of mutants affected in the production, transmission, perception and transduction of the systemic signal(s).     

RESPONSES OF PLANTS TO INFECTION BY BOTRYTIS CINEREA AND NOVEL MEANS INVOLVED IN REDUCING THEIR SUSCEPTIBILITY TO INFECTION

Once the inoculum of B. cinerea conies into contact with the host and starts to be active in the phyllosphere of a susceptible host tissue, a series of events take place. These events may develop into a process that leads to necrosis of the host, or may end in an arrested infection with minimal damage to the host tissue. Increased susceptibility to the pathogen is associated with factors that enhance ageing of the host tissues, such as the plant hormones ethylene and abscisic acid and elevation of free radical levels in the host tissue. Decreased susceptibility is obtained by inhibiting the production or activity of such factors in the presence of increased levels of plant hormones such as gibberellic acid, and by increasing the calcium content of the cell walls and by scavenging of free radicals in the host tissue. There is evidence for the induction of resistance in hosts affected by B. cinerea. Host tissues challenged by B. cinerea react at the DNA, RNA and protein level and accumulate pathogenicity related proteins, phytoalexins or other phenolic compounds. Deposition of polymers in cell walls and lignification have also been recorded in various hosts. The role of each of these factors in relation to protection is not clear. Moreover, some of the phenomena may occur too late to protect the host tissue against infection. Although the inhibition of specific proteins such as polygalacturonases has been suggested as a mechanism by which to inhibit disease, it is unlikely that the inhibition of one enzyme, would lead to significant restriction of infection. However, simultaneous inhibition of several hydrolytic enzymes produced by the pathogen should result in disease suppression. Possibilities of reducing the susceptibility of hosts or arresting further development of localized infections are discussed.     

Insect eggs induce a systemic acquired resistance in Arabidopsis

Although they constitute an inert stage of the insect's life, eggs trigger plant defences that lead to egg mortality or attraction of egg parasitoids. We recently found that salicylic acid (SA) accumulates in response to oviposition by the Large White butterfly Pieris brassicae, both in local and systemic leaves, and that plants activate a response that is similar to the recognition of pathogen‐associated molecular patterns (PAMPs), which are involved in PAMP‐triggered immunity (PTI). Here we discovered that natural oviposition by P. brassicae or treatment with egg extract inhibit growth of different Pseudomonas syringae strains in Arabidopsis through the activation of a systemic acquired resistance (SAR). This egg‐induced SAR involves the metabolic SAR signal pipecolic acid, depends on ALD1 and FMO1, and is accompanied by a stronger induction of defence genes upon secondary infection. Although P. brassicae larvae showed a reduced performance when feeding on Pseudomonas syringae‐infected plants, this effect was less pronounced when infected plants had been previously oviposited. Altogether, our results indicate that egg‐induced SAR might have evolved as a strategy to prevent the detrimental effect of bacterial pathogens on feeding larvae.     

Accumulation of gentisic acid as associated with systemic infections but not with the hypersensitive response in plant-pathogen interactions

In the present work we have studied the accumulation of gentisic acid (2,5-dihydroxybenzoic acid, a metabolic derivative of salicylic acid, SA) in the plant-pathogen systems, Cucumis sativus and Gynura aurantiaca, infected with either prunus necrotic ringspot virus (PNRSV) or the exocortis viroid (CEVd), respectively. Both pathogens produced systemic infections and accumulated large amounts of the intermediary signal molecule gentisic acid as ascertained by electrospray ionization mass spectrometry (ESI-MS) coupled on line with high performance liquid chromatography (HPLC). The compound was found mostly in a conjugated (β-glucoside) form. Gentisic acid has also been found to accumulate (although at lower levels) in cucumber inoculated with low doses of Pseudomonas syringae pv. tomato, producing a nonnecrotic reaction. In contrast, when cucumber was inoculated with high doses of this pathogen, a hypersensitive reaction occurred, but no gentisic-acid signal was induced. This is consistent with our results supporting the idea that gentisic-acid signaling may be restricted to nonnecrotizing reactions of the host plant (Bellés et al. in Mol Plant-Microbe Interact 12:227–235, 1999). In cucumber and Gynura plants, the activity of gentisic acid as inducing signal was different to that of SA, thus confirming the data found for tomato. Exogenously supplied gentisic acid was able to induce peroxidase activity in both Gynura and cucumber plants in a similar way as SA or pathogens. However, gentisic-acid treatments strongly induced polyphenol oxidase activity in cucumber, whereas pathogen infection or SA treatment resulted in a lower induction of this enzyme. Nevertheless, gentisic acid did not induce other defensive proteins which are induced by SA in these plants. This indicates that gentisic acid could act as an additional signal to SA for the activation of plant defenses in cucumber and Gynura plants.     

Aqueous fruit extracts of Azadirachta indica induce systemic acquired resistance in barley against Drechslera graminea

The study reported here primarily focuses on whether fruit extracts of Azadirachta indica Juss. (Neem) can induce systemic acquired resistance (SAR) in Hordeum vulgare against Drechslera graminea. A single leaf from each of the 1-month-old seedlings grown in 50 pots was treated with neem extract. Seven samples were collected at 12-h intervals for estimation of salicylic acid (SA) content and activities of phenylalanine ammonia lyase (PAL) and peroxidase (PO). Disease incidence was recorded on uninoculated leaves after 2 weeks and on newly emerged leaves after 3 weeks of inoculation of spores of the pathogen. Treatment of single leaf of barley seedlings with aqueous fruit extract of neem could protect the untreated and later emerging leaves of these seedlings from infection by leaf stripe pathogen. The concentration of SA and activities of PAL and PO were significantly higher in untreated leaves of seedlings given a single leaf treatment with neem fruit extract. The results show that neem fruit extract induced SAR in barley seedlings against D. graminea. The results of the study are significant for developing an environment-friendly biocide, which could induce SAR in crop plants leading to efficient management of pathogens     

A constitutive PR-1::luciferase expression screen identifies Arabidopsis mutants with differential disease resistance to both biotrophic and necrotrophic pathogens

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.