Design of a bacterial speck resistant tomato by CRISPR/Cas9‐mediated editing of SlJAZ2

Due to their different lifestyles, effective defence against biotrophic pathogens normally leads to increased susceptibility to necrotrophs, and vice versa. Solving this trade‐off is a major challenge for obtaining broad‐spectrum resistance in crops and requires uncoupling the antagonism between the jasmonate (JA) and salicylate (SA) defence pathways. Pseudomonas syringae pv. tomato (Pto) DC3000, the causal agent of tomato bacterial speck disease, produces coronatine (COR) that stimulates stomata opening and facilitates bacterial leaf colonization. In Arabidopsis, stomata response to COR requires the COR co‐receptor AtJAZ2, and dominant AtJAZ2Δjas repressors resistant to proteasomal degradation prevent stomatal opening by COR. Here, we report the generation of a tomato variety resistant to the bacterial speck disease caused by PtoDC3000 without compromising resistance to necrotrophs. We identified the functional ortholog of AtJAZ2 in tomato, found that preferentially accumulates in stomata and proved that SlJAZ2 is a major co‐receptor of COR in stomatal guard cells. SlJAZ2 was edited using CRISPR/Cas9 to generate dominant JAZ2 repressors lacking the C‐terminal Jas domain (SlJAZ2Δjas). SlJAZ2Δjas prevented stomatal reopening by COR and provided resistance to PtoDC3000. Water transpiration rate and resistance to the necrotrophic fungal pathogen Botrytis cinerea, causal agent of the tomato gray mold, remained unaltered in Sljaz2Δjas plants. Our results solve the defence trade‐off in a crop, by spatially uncoupling the SA‐JA hormonal antagonism at the stomata, entry gates of specific microbes such as PtoDC3000. Moreover, our results also constitute a novel CRISPR/Cas‐based strategy for crop protection that could be readily implemented in the field.     

Hyaluronic acid of Streptococcus sp. as a potent elicitor for induction of systemic resistance against plant diseases

The potential of hyaluronic acid (HA) in inducing systemic resistance to cucumber, tomato and pepper was tested in planta. In the study, HA was found to be a potent agent for suppressing disease caused by Cucumber Mosaic Virus (CMV) (in pepper), Pseudomonas syringae pv. tomato (tomato speck disease), Xanthomonas axonopodis pv. vesicatoria (tomato spot disease), Pseudomonas syringae pv. lachrymans (cucumber angular leaf spot), and Colletotrichum orbiculare (cucumber anthracnose). Disease control was obtained with spraying, injection and drenching of plants with HA. HA did not exhibit direct antimicrobial action against the pathogens tested. Studies carried out in transgenic tobacco indicated that defense genes PR 1a and PDF 1.2 were activated upon treatment with HA, demonstrating salicylic acid (SA) and jasmonic acid (JA) pathways getting activated during defense. Further work is warranted to evaluate the use of HA-mediated disease suppression in crop plants.     

Jasmonic acid and salicylic acid play minor roles in stomatal regulation by CO2, abscisic acid,darkness, vapor pressure deficit and ozone

Jasmonic acid (JA) and salicylic acid (SA) regulate stomatal closure, preventing pathogen invasion into plants. However, to what extent abscisic acid (ABA), SA and JA interact, and what the roles of SA and JA are in stomatal responses to environmental cues, remains unclear. Here, by using intact plant gas-exchange measurements in JA and SA single and double mutants, we show that stomatal responsiveness to CO₂, light intensity, ABA, high vapor pressure deficit and ozone either did not or, for some stimuli only, very slightly depended upon JA and SA biosynthesis and signaling mutants, including dde2, sid2, coi1, jai1, myc2 and npr1 alleles. Although the stomata in the mutants studied clearly responded to ABA, CO₂, light and ozone, ABA-triggered stomatal closure in npr1-1 was slightly accelerated compared with the wild type. Stomatal reopening after ozone pulses was quicker in the coi1-16 mutant than in the wild type. In intact Arabidopsis plants, spraying with methyl-JA led to only a modest reduction in stomatal conductance 80 min after treatment, whereas ABA and CO₂ induced pronounced stomatal closure within minutes. We could not document a reduction of stomatal conductance after spraying with SA. Coronatine-induced stomatal opening was initiated slowly after 1.5–2.0 h, and reached a maximum by 3 h after spraying intact plants. Our results suggest that ABA, CO₂ and light are major regulators of rapid guard cell signaling, whereas JA and SA could play only minor roles in the whole-plant stomatal response to environmental cues in Arabidopsis and Solanum lycopersicum (tomato).     

Expression of tomato salicylic acid (SA)‐responsive pathogenesis‐related genes in Mi‐1‐mediated and SA‐induced resistance to root‐knot nematodes

The expression pattern of pathogenesis‐related genes PR‐1, PR‐2 and PR‐5, considered as markers for salicylic acid (SA)‐dependent systemic acquired resistance (SAR), was examined in the roots and shoots of tomato plants pre‐treated with SA and subsequently infected with root‐knot nematodes (RKNs) (Meloidogyne incognita). PR‐1 was up‐regulated in both roots and shoots of SA‐treated plants, whereas the expression of PR‐5 was enhanced only in roots. The over‐expression of PR‐1 in the whole plant occurred as soon as 1 day after SA treatment. Up‐regulation of the PR‐1 gene was considered to be the main marker of SAR elicitation. One day after treatment, plants were inoculated with active juveniles (J2s) of M. incognita. The number of J2s that entered the roots and started to develop was significantly lower in SA‐treated than in untreated plants at 5 and 15 days after inoculation. The expression pattern of PR‐1, PR‐2 and PR‐5 was also examined in the roots and shoots of susceptible and Mi‐1‐carrying resistant tomato plants infected by RKNs. Nematode infection produced a down‐regulation of PR genes in both roots and shoots of SA‐treated and untreated plants, and in roots of Mi‐carrying resistant plants. Moreover, in resistant infected plants, PR gene expression, in particular PR‐1 gene expression, was highly induced in shoots. Thus, nematode infection was demonstrated to elicit SAR in shoots of resistant plants. The data presented in this study show that the repression of host defence SA signalling is associated with the successful development of RKNs, and that SA exogenously added as a soil drench is able to trigger a SAR‐like response to RKNs in tomato.     

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.     

Different mechanisms of Trichoderma virens‐mediated resistance in tomato against Fusarium wilt involve the jasmonic and salicylic acid pathways

In the present study, we investigated the role of Trichoderma virens (TriV_JSB100) spores or cell‐free culture filtrate in the regulation of growth and activation of the defence responses of tomato (Solanum lycopersicum) plants against Fusarium oxysporum f. sp. lycopersici by the development of a biocontrol–plant–pathogen interaction system. Two‐week‐old tomato seedlings primed with TriV_JSB100 spores cultured on barley grains (BGS) or with cell‐free culture filtrate (CF) were inoculated with Fusarium pathogen under glasshouse conditions; this resulted in significantly lower disease incidence in tomato Oogata‐Fukuju plants treated with BGS than in those treated with CF. To dissect the pathways associated with this response, jasmonic acid (JA) and salicylic acid (SA) signalling in BGS‐ and CF‐induced resistance was evaluated using JA‐ and SA‐impaired tomato lines. We observed that JA‐deficient mutant def1 plants were susceptible to Fusarium pathogen when they were treated with BGS. However, wild‐type (WT) BGS‐treated tomato plants showed a higher JA level and significantly lower disease incidence. SA‐deficient mutant NahG plants treated with CF were also found to be susceptible to Fusarium pathogen and displayed low SA levels, whereas WT CF‐treated tomato plants exhibited moderately lower disease levels and substantially higher SA levels. Expression of the JA‐responsive defensin gene PDF1 was induced in WT tomato plants treated with BGS, whereas the SA‐inducible pathogenesis‐related protein 1 acidic (PR1a) gene was up‐regulated in WT tomato plants treated with CF. These results suggest that TriV_JSB100 BGS and CF differentially induce JA and SA signalling cascades for the elicitation of Fusarium oxysporum resistance in tomato.     

Hexanoic acid protects tomato plants against Botrytis cinerea by priming defence responses and reducing oxidative stress

Treatment with the resistance priming inducer hexanoic acid (Hx) protects tomato plants from Botrytis cinerea by activating defence responses. To investigate the molecular mechanisms underlying hexanoic acid‐induced resistance (Hx‐IR), we compared the expression profiles of three different conditions: Botrytis‐infected plants (Inf), Hx‐treated plants (Hx) and Hx‐treated + infected plants (Hx+Inf). The microarray analysis at 24 h post‐inoculation showed that Hx and Hx+Inf plants exhibited the differential expression and priming of many Botrytis‐induced genes. Interestingly, we found that the activation by Hx of other genes was not altered by the fungus at this time point. These genes may be considered to be specific targets of the Hx priming effect and may help to elucidate its mechanisms of action. It is noteworthy that, in Hx and Hx+Inf plants, there was up‐regulation of proteinase inhibitor genes, DNA‐binding factors, enzymes involved in plant hormone signalling and synthesis, and, remarkably, the genes involved in oxidative stress. Given the relevance of the oxidative burst occurring in plant–pathogen interactions, the effect of Hx on this process was studied in depth. We showed by specific staining that reactive oxygen species (ROS) accumulation in Hx+Inf plants was reduced and more restricted around infection sites. In addition, these plants showed higher ratios of reduced to oxidized glutathione and ascorbate, and normal levels of antioxidant activities. The results obtained indicate that Hx protects tomato plants from B. cinerea by regulating and priming Botrytis‐specific and non‐specific genes, preventing the harmful effects of oxidative stress produced by infection.     

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.     

Potential of salicylic acid activity derived from stress-induced (water) Tomato against Meloidogyne incognita

Plants have evolved several types of sophisticated defence mechanisms to protect themselves from enemies in which the signalling molecules such as salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) often play crucial roles. An experiment was conducted to investigate the water induced stress in young tomato plants (Lycopersicon esculentum var. Pusa Ruby) during water deprivation and after relief from stress in association with salicylic acid. Application of SA or certain analogues such as aspirin induces expression of pathogenesis-related proteins (PR) in plants which serve as molecular markers for the systemic acquired response (SAR). In plants, the production of reactive oxygen species (ROS), such as superoxide radical (O₂), hydroxyl radical (OH) and hydrogen peroxide (H₂O₂), is also common in metabolic and physiological processes. Similar to the defence-regulating compounds such as SA and JA, plant-derived ET is also known to be involved in disease resistance. The role of ROS or ET could be investigated which was carried out in the present study to know the osmotic stress mediated resistance by SA against Meloidogyne incognita in tomato plants.     

Chitosan signaling in guard cells requires endogenous salicylic acid

An elicitor chitosan (CHT) induces stomatal closure but the mechanism remains to be clarified. A phytohormone salicylic acid (SA) is crucial for elicitor-induced defense signaling in plants. Here we investigated whether endogenous SA is required for CHT signaling in guard cells. In the SA-deficient nahG mutant, treatment of CHT did not induce either apoplastic reactive oxygen species (ROS) production or stomatal closure but co-treatment of CHT and SA induced both apoplastic ROS production and stomatal closure, indicating the involvement of endogenous SA in CHT-induced apoplastic ROS production and CHT-induced stomatal closure. Furthermore, CHT induced transient cytosolic free calcium concentration increments in the nahG mutant in the presence of exogenous SA but not in the absence of exogenous SA. These results provide evidence that endogenous SA is a crucial element in CHT-induced stomatal closure.     

Involvement of salicylic acid in induction of nematode resistance in plants

The role of salicylic acid (SA) as a possible signaling component in the case of the infection of plants with nematodes has been studied using a model system consisting of the tomato (Lycopersicon esculentum (Mill.) and race 1 of the gall eelworm Meloidogyne incognita (Kofoid and White, 1919; Chitwood, 1949). The preplanting SA treatment of tomato seeds results in an increased nematode resistance of susceptible tomato cultivars; the protective effect is higher in the case of SA combined with chitosan, a biogenic elicitor of plant resistance. The studied preparations stimulate the growth and development of the plants. The increase in the resistance of tomato plants is related to the increased activity of phenylalanine ammonia-lyase and an increased SA content in plant tissues infected with nematodes; both these factors significantly influence nematode development.     

Influence of salicylic acid and Glomus fasciculatum on fusarial wilt of tomato and brinjal

Wilt of tomato caused by Fusarium oxysporum f.sp. lycopersici and wilt of brinjal caused by Fusarium solani are very common in different agricultural fields of West Bengal, India. The study on the effect of salicylic acid (SA) on in vitro growth of the pathogens revealed that SA completely checks the growth at 0.7 mM concentration and above. On the other hand, the percentage of mycorrhisation in the host plants with an arbuscular mycorrhizal (AM) fungus, Glomus fasciculatum was found to be decreased in the presence of SA in the plants with or without pathogenic infection. Treatment of tomato and brinjal plants either singly with AM fungus or with SA (0.5 and 1.0 mM) and also with their combined treatment showed amelioration of plant height, length of root, fresh weight of root and fresh weight of plants. However, the AM fungus-treated plants showed highest growth responses. The result also reveals that integrated treatment with AM plus SA has significant effect on reduction of infection where application of AM + SA (1.0 mM) resulted in 67.16 and 69.70% reduction of fusarial wilt infection in tomato and brinjal plants, respectively.     

Priming for enhanced defence responses by specific inhibition of the Arabidopsis response to coronatine

The priming agent β-aminobutyric acid (BABA) is known to enhance Arabidopsis resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 by potentiating salicylic acid (SA) defence signalling, notably PR1 expression. The molecular mechanisms underlying this phenomenon remain unknown. A genome-wide microarray analysis of BABA priming during Pst DC3000 infection revealed direct and primed up-regulation of genes that are responsive to SA, the SA analogue benzothiadiazole and pathogens. In addition, BABA was found to inhibit the Arabidopsis response to the bacterial effector coronatine (COR). COR is known to promote bacterial virulence by inducing the jasmonic acid (JA) response to antagonize SA signalling activation. BABA specifically repressed the JA response induced by COR without affecting other plant JA responses. This repression was largely SA-independent, suggesting that it is not caused by negative cross-talk between SA and JA signalling cascades. Treatment with relatively high concentrations of purified COR counteracted BABA inhibition. Under these conditions, BABA failed to protect Arabidopsis against Pst DC3000. BABA did not induce priming and resistance in plants inoculated with a COR-deficient strain of Pst DC3000 or in the COR-insensitive mutant coi1-16. In addition, BABA blocked the COR-dependent re-opening of stomata during Pst DC3000 infection. Our data suggest that BABA primes for enhanced resistance to Pst DC3000 by interfering with the bacterial suppression of Arabidopsis SA-dependent defences. This study also suggests the existence of a signalling node that distinguishes COR from other JA responses.     

The Bacterial Effector HopX1 Targets JAZ Transcriptional Repressors to Activate Jasmonate Signaling and Promote Infection in Arabidopsis

Pathogenicity of Pseudomonas syringae is dependent on a type III secretion system, which secretes a suite of virulence effector proteins into the host cytoplasm, and the production of a number of toxins such as coronatine (COR), which is a mimic of the plant hormone jasmonate-isoleuce (JA-Ile). Inside the plant cell, effectors target host molecules to subvert the host cell physiology and disrupt defenses. However, despite the fact that elucidating effector action is essential to understanding bacterial pathogenesis, the molecular function and host targets of the vast majority of effectors remain largely unknown. Here, we found that effector HopX1 from Pseudomonas syringae pv. tabaci (Pta) 11528, a strain that does not produce COR, interacts with and promotes the degradation of JAZ proteins, a key family of JA-repressors. We show that hopX1 encodes a cysteine protease, activity that is required for degradation of JAZs by HopX1. HopX1 associates with JAZ proteins through its central ZIM domain and degradation occurs in a COI1-independent manner. Moreover, ectopic expression of HopX1 in Arabidopsis induces the expression of JA-dependent genes, represses salicylic acid (SA)-induced markers, and complements the growth of a COR-deficient P. syringae pv. tomato (Pto) DC3000 strain during natural bacterial infections. Furthermore, HopX1 promoted susceptibility when delivered by the natural type III secretion system, to a similar extent as the addition of COR, and this effect was dependent on its catalytic activity. Altogether, our results indicate that JAZ proteins are direct targets of bacterial effectors to promote activation of JA-induced defenses and susceptibility in Arabidopsis. HopX1 illustrates a paradigm of an alternative evolutionary solution to COR with similar physiological outcome.