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.     

Host‐induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce

Host‐induced gene silencing (HIGS) is an RNA interference‐based approach in which small interfering RNAs (siRNAs) are produced in the host plant and subsequently move into the pathogen to silence pathogen genes. As a proof‐of‐concept, we generated stable transgenic lettuce plants expressing siRNAs targeting potentially vital genes of Bremia lactucae, a biotrophic oomycete that causes downy mildew, the most important disease of lettuce worldwide. Transgenic plants, expressing inverted repeats of fragments of either the Highly Abundant Message #34 (HAM34) or Cellulose Synthase (CES1) genes of B. lactucae, specifically suppressed expression of these genes, resulting in greatly reduced growth and inhibition of sporulation of B. lactucae. This demonstrates that HIGS can provide effective control of B. lactucae in lettuce; such control does not rely on ephemeral resistance conferred by major resistance genes and therefore offers new opportunities for durable control of diverse diseases in numerous crops.     

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.     

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.     

The toolbox of Trichoderma spp. in the biocontrol of Botrytis cinerea disease

Botrytis cinerea is a necrotrophic fungal pathogen causing disease in many plant species, leading to economically important crop losses. So far, fungicides have been widely used to control this pathogen. However, in addition to their detrimental effects on the environment and potential risks for human health, increasing fungicide resistance has been observed in the B. cinerea population. Biological control, that is the application of microbial organisms to reduce disease, has gained importance as an alternative or complementary approach to fungicides. In this respect, the genus Trichoderma constitutes a promising pool of organisms with potential for B. cinerea control. In the first part of this article, we review the specific mechanisms involved in the direct interaction between the two fungi, including mycoparasitism, the production of antimicrobial compounds and enzymes (collectively called antagonism), and competition for nutrients and space. In addition, biocontrol has also been observed when Trichoderma is physically separated from the pathogen, thus implying an indirect systemic plant defence response. Therefore, in the second part, we describe the consecutive steps leading to induced systemic resistance (ISR), starting with the initial Trichoderma–plant interaction and followed by the activation of downstream signal transduction pathways and, ultimately, the defence response resulting in ISR (ISR‐prime phase). Finally, we discuss the ISR‐boost phase, representing the effect of ISR priming by Trichoderma spp. on plant responses after additional challenge with B. cinerea.     

The biotrophy-associated secreted protein 4 (BAS4) participates in the transition of Magnaporthe oryzae from the biotrophic to the necrotrophic phase

The physiological and metabolic processes of host plants are manipulated and remodeled by phytopathogenic fungi during infection, revealed obvious signs of biotrophy of the hemibiotrophic pathogen. As we known that effector proteins play key roles in interaction of hemibiotrophic fungi and their host plants. BAS4 (biotrophy-associated secreted protein 4) is an EIHM (extrainvasive hyphal membrane) matrix protein that was highly expressed in infectious hyphae. In order to study whether BAS4 is involved in the transition of rice blast fungus from biotrophic to necrotrophic phase, The susceptible rice cultivar Lijiangxintuanheigu (LTH) that were pre-treated with prokaryotic expression product of BAS4 and then followed with inoculation of the blast strain, more serious blast disease symptom, more biomass such as sporulation and fungal relative growth, and lower expression level of pathogenicity-related genes appeared in lesion of the rice leaves than those of the PBS-pretreated-leaves followed with inoculation of the same blast strain, which demonstrating that BAS4 invitro changed rice defense system to facilitate infection of rice blast strain. And the susceptible rice cultivar (LTH) were inoculated withBAS4-overexpressed blast strain, we also found more serious blast disease symptom and more biomass also appeared in lesion of leaves inoculated with BAS4-overexpressed strain than those of leaves inoculated with the wild-type strain, and expression level of pathogenicity-related genes appeared lower in biotrophic phase and higher in necrotrophic phase of infection, indicating BAS4 maybe in vivo regulate defense system of rice to facilitate transition of biotrophic to necrotrophic phase. Our data demonstrates that BAS4 in vitro and in vivo participates in transition from the biotrophic to the necrotrophic phase of Magnaporthe oryzae.     

Tryptophan‐derived secondary metabolites in Arabidopsis thaliana confer non‐host resistance to necrotrophic Plectosphaerella cucumerina fungi

A defence pathway contributing to non‐host resistance to biotrophic fungi in Arabidopsis involves the synthesis and targeted delivery of the tryptophan (trp)‐derived metabolites indol glucosinolates (IGs) and camalexin at pathogen contact sites. We have examined whether these metabolites are also rate‐limiting for colonization by necrotrophic fungi. Inoculation of Arabidopsis with adapted or non‐adapted isolates of the ascomycete Plectosphaerella cucumerina triggers the accumulation of trp‐derived metabolites. We found that their depletion in cyp79B2 cyp79B3 mutants renders Arabidopsis fully susceptible to each of three tested non‐adapted P. cucumerina isolates, and super‐susceptible to an adapted P. cucumerina isolate. This assigns a key role to trp‐derived secondary metabolites in limiting the growth of both non‐adapted and adapted necrotrophic fungi. However, 4‐methoxy‐indol‐3‐ylmethylglucosinolate, which is generated by the P450 monooxygenase CYP81F2, and hydrolyzed by PEN2 myrosinase, together with the antimicrobial camalexin play a minor role in restricting the growth of the non‐adapted necrotrophs. This contrasts with a major role of these two trp‐derived phytochemicals in limiting invasive growth of non‐adapted biotrophic powdery mildew fungi, thereby implying the existence of other unknown trp‐derived metabolites in resistance responses to non‐adapted necrotrophic P. cucumerina. Impaired defence to non‐adapted P. cucumerina, but not to the non‐adapted biotrophic fungus Erysiphe pisi, on cyp79B2 cyp79B3 plants is largely restored in the irx1 background, which shows a constitutive accumulation of antimicrobial peptides. Our findings imply differential contributions of antimicrobials in non‐host resistance to necrotrophic and biotrophic pathogens.     

Cyclic lipopeptides from Bacillus subtilis activate distinct patterns of defence responses in grapevine

Non‐self‐recognition of microorganisms partly relies on the perception of microbe‐associated molecular patterns (MAMPs) and leads to the activation of an innate immune response. Bacillus subtilis produces three main families of cyclic lipopeptides (LPs), namely surfactins, iturins and fengycins. Although LPs are involved in induced systemic resistance (ISR) activation, little is known about defence responses induced by these molecules and their involvement in local resistance to fungi. Here, we showed that purified surfactin, mycosubtilin (iturin family) and plipastatin (fengycin family) are perceived by grapevine plant cells. Although surfactin and mycosubtilin stimulated grapevine innate immune responses, they differentially activated early signalling pathways and defence gene expression. By contrast, plipastatin perception by grapevine cells only resulted in early signalling activation. Gene expression analysis suggested that mycosubtilin activated salicylic acid (SA) and jasmonic acid (JA) signalling pathways, whereas surfactin mainly induced an SA‐regulated response. Although mycosubtilin and plipastatin displayed direct antifungal activity, only surfactin and mycosubtilin treatments resulted in a local long‐lasting enhanced tolerance to the necrotrophic fungus Botrytis cinerea in grapevine leaves. Moreover, challenge with specific strains overproducing surfactin and mycosubtilin led to a slightly enhanced stimulation of the defence response compared with the LP‐non‐producing strain of B. subtilis. Altogether, our results provide the first comprehensive view of the involvement of LPs from B. subtilis in grapevine plant defence and local resistance against the necrotrophic pathogen Bo. cinerea. Moreover, this work is the first to highlight the ability of mycosubtilin to trigger an immune response in plants.     

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.