Infection of Arabidopsis with a necrotrophic pathogen,Botrytis cinerea,elicits various defense responses but does not induce systemic acquired resistance (SAR)

Botrytis cinerea is a non-specific necrotrophic pathogen that attacks more than 200 plant species. In contrast to biotrophs, the necrotrophs obtain their nutrients by first killing the host cells. Many studies have shown that infection of plants by necrosis-causing pathogens induces a systemic acquired resistance (SAR), which provides protection against successive infections by a range of pathogenic organisms. We analyzed the role of SAR in B. cinerea infection of Arabidopsis. We show that although B. cinerea induced necrotic lesions and camalexin biosynthesis, it did not induce SAR-mediated protection against virulent strains of Pseudomonas syringae, or against subsequent B. cinerea infections. Induction of SAR with avirulent P. syringae or by chemical treatment with salicylic acid (SA) or benzothiadiazole also failed to inhibit B. cinerea growth, although removal of basal SA accumulation by expression of a bacterial salicylate hydroxylase (NahG) gene or by infiltration of 2-aminoindan-2-phosphonic acid, an inhibitor of phenylpropanoid pathway, increased B. cinerea disease symptoms. In addition, we show that B. cinerea induced expression of genes associated with SAR, general stress and ethylene/jasmonate-mediated defense pathways. Thus, B. cinerea does not induce SAR nor is it affected by SAR, making it a rare example of a necrogenic pathogen that does not cause SAR.     

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.     

Plant defense response against Fusarium oxysporum and strategies to develop tolerant genotypes in banana

Soil-borne fungal pathogen, Fusarium oxysporum causes major economic losses by inducing necrosis and wilting symptoms in many crop plants. Management of fusarium wilt is achieved mainly by the use of chemical fungicides which affect the soil health and their efficiency is often limited by pathogenic variability. Hence understanding the nature of interaction between pathogen and host may help to select and improve better cultivars. Current research evidences highlight the role of oxidative burst and antioxidant enzymes indicating that ROS act as an important signaling molecule in banana defense response against Fusarium oxysporum f.sp. cubense. The role of jasmonic acid signaling in plant defense against necrotrophic pathogens is well recognized. But recent studies show that the role of salicylic acid is complex and ambiguous against necrotrophic pathogens like Fusarium oxysporum, leading to many intriguing questions about its relationship between other signaling compounds. In case of banana, a major challenge is to identify specific receptors for effector proteins like SIX proteins and also the components of various signal transduction pathways. Significant progress has been made to uncover the role of defense genes but is limited to only model plants such as Arabidopsis and tomato. Keeping this in view, we review the host response, pathogen diversity, current understanding of biochemical and molecular changes that occur during host and pathogen interaction. Developing resistant cultivars through mutation, breeding, transgenic and cisgenic approaches have been discussed. This would help us to understand host defenses against Fusarium oxysporum and to formulate strategies to develop tolerant cultivars.     

Raphanusanin-mediated resistance to pathogens is light dependent in radish and Arabidopsis thaliana

Raphanusanin (Ra) is a light-induced inhibitor of hypocotyl growth that responds to unilateral blue light illumination in radish seedlings. We have previously shown that Ra regulates genes that are involved in common defense mechanisms. Many genes that are induced by Ra are also positively regulated by early blue light. To extend the understanding of the role of Ra in pathogen defense, we evaluated the effects of Ra on radish and Arabidopsis thaliana (A. thaliana) infected with the necrotrophic pathogen Botrytis cinerea (B. cinerea) and biotrophic pathogen Pseudomonas syringae (P. syringae). Radish and A. thaliana were found to be resistant to both pathogens when treated with Ra, depending on the concentration used. Interestingly, Ra-mediated resistance to P. syringae is dependent on light because Ra-treated seedlings exhibited enhanced susceptibility to P. syringae infection when grown in the dark. In addition to regulating the biotic defense response, Ra inhibited seed germination and root elongation and enhanced the growth of root hairs in the presence of light in radish and A. thaliana. Our data suggest that Ra regulates the expression of a set of genes involved in defense signaling pathways and plays a role in pathogen defense and plant development. Our results show that light may be generally required not only for the accumulation of Ra but also for its activation during the pathogen defense response.     

Influence of over-expression of cytosolic aspartate aminotransferase on amino acid metabolism and defence responses against Botrytis cinerea infection in Arabidopsis thaliana

Arabidopsis possesses several genes encoding aspartate aminotransferase, which catalyzes the bidirectional conversion of aspartate into glutamate. These amino acids together with asparagine and glutamine play an important role in N storage and distribution. In addition, they act as precursors for other amino acids. The gene encoding cytosolic aspartate aminotransferase, Asp2, was found to be induced upon infection with the necrotrophic pathogen Botrytis cinerea in Arabidopsis. Asp2 over-expression lines and a T-DNA insertion mutant were used to study the role of aspartate aminotransferase in Arabidopsis defence responses. Over-expression of Asp2 led to changes in aspartate content and aspartate-derived amino acids. The Asp2 knockout mutant was also slightly affected in its amino acid composition. Under standard growth conditions, the Asp2 transgenic lines did not show morphological changes in comparison with the wild-type. However, transgenic lines with the highest Asp2 expression displayed more spreading lesions when infected with B. cinerea. We discuss how this gene involved in amino acid metabolism might interact with plant defence responses.     

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.     

Cell wall-localized BETA-XYLOSIDASE4 contributes to immunity of Arabidopsis against Botrytis cinerea

Plant cell walls constitute physical barriers that restrict access of microbial pathogens to the contents of plant cells. The primary cell wall of multicellular plants predominantly consists of cellulose, hemicellulose, and pectin, and its composition can change upon stress. BETA-XYLOSIDASE4 (BXL4) belongs to a seven-member gene family in Arabidopsis (Arabidopsis thaliana), one of which encodes a protein (BXL1) involved in cell wall remodeling. We assayed the influence of BXL4 on plant immunity and investigated the subcellular localization and enzymatic activity of BXL4, making use of mutant and overexpression lines. BXL4 localized to the apoplast and was induced upon infection with the necrotrophic fungal pathogen Botrytis cinerea in a jasmonoyl isoleucine-dependent manner. The bxl4 mutants showed a reduced resistance to B. cinerea, while resistance was increased in conditional overexpression lines. Ectopic expression of BXL4 in Arabidopsis seed coat epidermal cells rescued a bxl1 mutant phenotype, suggesting that, like BXL1, BXL4 has both xylosidase and arabinosidase activity. We conclude that BXL4 is a xylosidase/arabinosidase that is secreted to the apoplast and its expression is upregulated under pathogen attack, contributing to immunity against B. cinerea, possibly by removal of arabinose and xylose side-chains of polysaccharides in the primary cell wall.

A putative bifunctional xylosidase/arabinofuranisodase localizes to the apoplast and is important for immunity against the necrotrophic pathogen Botrytis cinerea.     

A mutation in the expansin‐like A2 gene enhances resistance to necrotrophic fungi and hypersensitivity to abiotic stress in Arabidopsis thaliana

Expansins are cell wall loosening agents, known for their endogenous function in cell wall extensibility. The Arabidopsis expansin‐like A2 (EXLA2) gene was identified by its down‐regulation in response to infection by the necrotrophic pathogen Botrytis cinerea, and by the reduced susceptibility of an exla2 mutant to the same pathogen. The exla2 mutant was equally susceptible to Pseudomonas syringae pv. tomato, but was more resistant to the necrotrophic fungus Alternaria brassicicola, when compared with the wild‐type or with transgenic, ectopic EXLA2‐overexpressing lines. The exla2 mutants also enhanced tolerance to the phytoprostane‐A₁. This suggests that the absence or down‐regulation of EXLA2 leads to increased resistance to B. cinerea in a CORONATINE INSENSITIVE 1 (COI1)‐dependent manner, and this down‐regulation can be achieved by phytoprostane‐A₁ treatment. EXLA2 is induced significantly by salinity and cold, and by the exogenous application of abscisic acid. The exla2 mutant also showed hypersensitivity towards increased salt and cold, and this hypersensitivity required a functional abscisic acid pathway. The differential temporal expression of EXLA2 and the phenotypes in transgenic plants with altered expression of EXLA2 indicate that plant cell wall structure is an important player during Arabidopsis developmental stages. Our results indicate that EXLA2 appears to be important in response to various biotic and abiotic stresses, particularly in the pathogenesis of necrotrophic pathogens and in the tolerance to abiotic stress.     

Silencing of OPR3 in tomato reveals the role of OPDA in callose deposition during the activation of defense responses against Botrytis cinerea

Cis‐(+)‐12‐oxo‐phytodienoic acid (OPDA) is likely to play signaling roles in plant defense that do not depend on its further conversion to the phytohormone jasmonic acid. To elucidate the role of OPDA in Solanum lycopersicum (tomato) plant defense, we have silenced the 12‐oxophytodienoate reductase 3 (OPR3) gene. Two independent transgenic tomato lines (SiOPR3‐1 and SiOPR3‐2) showed significantly reduced OPR3 expression upon infection with the necrotrophic pathogen Botrytis cinerea. Moreover, SiOPR3 plants are more susceptible to this pathogen, and this susceptibility is accompanied by a significant decrease in OPDA levels and by the production of JA‐Ile being almost abolished. OPR3 silencing also leads to a major reduction in the expression of other genes of the jasmonic acid (JA) synthesis and signaling pathways after infection. These results confirm that in tomato plants, as in Arabidopsis, OPR3 determines OPDA availability for JA biosynthesis. In addition, we show that an intact JA biosynthetic pathway is required for proper callose deposition, as its pathogen‐induced accumulation is reduced in SiOPR3 plants. Interestingly, OPDA, but not JA, treatment restored basal resistance to B. cinerea and induced callose deposition in SiOPR3‐1 and SiOPR3‐2 transgenic plants. These results provide clear evidence that OPDA by itself plays a major role in the basal defense of tomato plants against this necrotrophic pathogen.     

Reactive oxygen species generated in chloroplasts contribute to tobacco leaf infection by the necrotrophic fungus Botrytis cinerea

Reactive oxygen species (ROS) play fundamental roles in plant responses to pathogen infection, including modulation of cell death processes and defense‐related gene expression. Cell death triggered as part of the hypersensitive response enhances resistance to biotrophic pathogens, but favors the virulence of necrotrophs. Even though the involvement of ROS in the orchestration of defense responses is well established, the relative contribution of specific subcellular ROS sources to plant resistance against microorganisms with different pathogenesis strategies is not completely known. The aim of this work was to investigate the role of chloroplastic ROS in plant defense against a typical necrotrophic fungus, Botrytis cinerea. For this purpose, we used transgenic Nicotiana tabacum (tobacco) lines expressing a plastid‐targeted cyanobacterial flavodoxin (pfld lines), which accumulate lower chloroplastic ROS in response to different stresses. Tissue damage and fungal growth were significantly reduced in infected leaves of pfld plants, as compared with infected wild‐type (WT) counterparts. ROS build‐up triggered by Botrytis infection and associated with chloroplasts was significantly decreased (70–80%) in pfld leaves relative to the wild type. Phytoalexin accumulation and expression of pathogenesis‐related genes were induced to a lower degree in pfld plants than in WT siblings. The impact of fungal infection on photosynthetic activity was also lower in pfld leaves. The results indicate that chloroplast‐generated ROS play a major role in lesion development during Botrytis infection. This work demonstrates that the modulation of chloroplastic ROS levels by the expression of a heterologous antioxidant protein can provide a significant degree of protection against a canonical necrotrophic fungus.