Induce Plant Defense Responses Research Articles

The Botrytis cinerea Xylanase BcXyl1 Modulates Plant Immunity.

Botrytis cinerea is one of the most notorious pathogenic species that causes serious plant diseases and substantial losses in agriculture throughout the world. We identified BcXyl1 from B. cinerea that exhibited xylanase activity. Expression of the BcXyl1 gene was strongly induced in B. cinerea infecting Nicotiana benthamiana and tomato plants, and BcXyl1 deletion strains severely compromised the virulence of B. cinerea. BcXyl1 induced strong cell death in several plants, and cell death activity of BcXyl1 was independent of its xylanase activity. Purified BcXyl1 triggered typically PAMP-triggered immunity (PTI) responses and conferred resistance to B. cinerea and TMV in tobacco and tomato plants. A 26-amino acid peptide of BcXyl1 was sufficient for elicitor function. Furthermore, the BcXyl1 death-inducing signal was mediated by the plant LRR receptor-like kinases (RLKs) BAK1 and SOBIR1. Our data suggested that BcXyl1 contributed to B. cinerea virulence and induced plant defense responses.

Distinct Signatures of Host Defense Suppression by Plant-Feeding Mites.

Tomato plants are attacked by diverse herbivorous arthropods, including by cell-content-feeding mites, such as the extreme generalist Tetranychus urticae and specialists like Tetranychus evansi and Aculops lycopersici. Mite feeding induces plant defense responses that reduce mite performance. However, T. evansi and A. lycopersici suppress plant defenses via poorly understood mechanisms and, consequently, maintain a high performance on tomato. On a shared host, T. urticae can be facilitated by either of the specialist mites, likely due to the suppression of plant defenses. To better understand defense suppression and indirect plant-mediated interactions between herbivorous mites, we used gene-expression microarrays to analyze the transcriptomic changes in tomato after attack by either a single mite species (T. urticae, T. evansi, A. lycopersici) or two species simultaneously (T. urticae plus T. evansi or T. urticae plus A. lycopersici). Additionally, we assessed mite-induced changes in defense-associated phytohormones using LC-MS/MS. Compared to non-infested controls, jasmonates (JAs) and salicylate (SA) accumulated to higher amounts upon all mite-infestation treatments, but the response was attenuated after single infestations with defense-suppressors. Strikingly, whereas 8 to 10% of tomato genes were differentially expressed upon single infestations with T. urticae or A. lycopersici, respectively, only 0.1% was altered in T. evansi-infested plants. Transcriptome analysis of dual-infested leaves revealed that A. lycopersici primarily suppressed T. urticae-induced JA defenses, while T. evansi dampened T. urticae-triggered host responses on a transcriptome-wide scale. The latter suggests that T. evansi not solely down-regulates plant gene expression, but rather directs it back towards housekeeping levels. Our results provide valuable new insights into the mechanisms underlying host defense suppression and the plant-mediated facilitation of competing herbivores.

A Verticillium dahliae Pectate Lyase Induces Plant Immune Responses and Contributes to Virulence.

Verticillium dahliae is a wide-host-range fungal pathogen that causes soil-borne disease in hundreds of dicotyledonous hosts. In search of V. dahliae Vd991 cell death-inducing proteins, we identified a pectate lyase (VdPEL1) that exhibited pectin hydrolytic activity, which could induce strong cell death in several plants. Purified VdPEL1 triggered defense responses and conferred resistance to Botrytis cinerea and V. dahliae in tobacco and cotton plants. Our results demonstrated that the mutant VdPEL1rec lacking the enzymatic activity lacked functions to induce both cell death and plant resistance, implying that the enzymatic activity was necessary. In addition, VdPEL1 was strongly induced in V. dahliae infected Nicotiana benthamiana and cotton roots, and VdPEL1 deletion strains severely compromised the virulence of V. dahliae. Our data suggested that VdPEL1 contributed to V. dahliae virulence and induced plant defense responses. These findings provide a new insight for the function of pectate lyase in the host–pathogen interaction.

Antibiotic producing endophytic Streptomyces spp. colonize above-ground plant parts and promote shoot growth in multiple healthy and pathogen-challenged cereal crops

The Streptomyces spp. used in this work were previously isolated as diazotrophic endophytes from sorghum stems. Here, we characterized the Streptomyces spp. for their colonization ability, plant growth promotion and protection against fungal disease in three cereals. In vitro analysis by dual culture study showed inhibitory effect on the rice pathogen Magnaporthe oryzae B157 along with inhibition of the ubiquitous phytopathogen Rhizoctonia solani by the Streptomyces spp. used in this study. The active compounds responsible for phytopathogen inhibition were extracted with ethyl acetate and tested positive against the fungal pathogens. GC–MS based identification of the active compounds responsible for fungal pathogen inhibition showed them to be 2-(chloromethyl)-2-cyclopropyloxirane, 2, 4- ditert-butylphenol and 1‐ethylthio‐3‐methyl‐1, 3‐butadiene in extracts of culture supernatants from the three different strains respectively. EGFP tagged Streptomyces strains showed profuse colonization in roots as well as aerial parts of cereal plants. Direct inhibitory action against M. oryzae B157 and R. solani correlated with the observation that upon fungal pathogen challenge, the bacterized rice, sorghum and wheat plants showed significantly good plant growth, particularly in aerial parts as compared to unbacterized controls. In addition, benefit was seen in inoculated healthy plants in terms of increase in wet weight of roots and shoots as compared to the uninoculated controls. The mechanism of biocontrol also involved induction of plant defense response as evidenced by the upregulation of PR10a, NPR1, PAL and LOX2 in Streptomyces colonized plants.

Functional analysis of eliciting plant response protein Epl1-Tas from Trichoderma asperellum ACCC30536

Eliciting plant response protein (Epl) is a small Trichoderma secreted protein that acts as an elicitor to induce plant defense responses against pathogens. In the present study, the differential expression, promoter analysis, and phylogenetic tree analysis of Epl1-Tas (GenBank JN966996) from T. asperellum ACCC30536 were performed. The results showed Epl1-Tas could play an important role in the interaction between T. asperellum ACCC30536 and woody plant or woody plant pathogen. Furthermore, the effect of the Escherichia coli recombinant protein rEpl1-e and the Pichia pastoris recombinant protein rEpl1-p on Populus davidiana × P. alba var. pyramidalis (PdPap) was studied. In PdPap seedlings, rEpl1-e or rEpl1-p induction altered the expression levels of 11 genes in the salicylic acid (SA, three genes), jasmonic acid (JA, four genes) and auxin (four genes) signal transduction pathways, and five kinds of enzymes activities The induction level of rEpl1-p was significantly higher than that of rEpl1-e, indicating that rEpl1-p could be used for further induction experiment. Under 3 mg/mL rEpl1-p induction, the mean height of the PdPap seedlings increased by 57.65% and the mean lesion area on the PdPap seedlings leaves challenged with Alternaria alternata decreased by 91.22% compared with those of the control. Thus, elicitor Epl1-Tas could induce the woody plant resistance to pathogen.

Survival and interaction of Escherichia coli O104:H4 on Arabidopsis thaliana and lettuce (Lactuca sativa) in comparison to E. coli O157:H7: Influence of plant defense response and bacterial capsular polysaccharide

Shiga toxin-producing Escherichia coli (STEC) has been associated with illnesses and outbreaks linked to fresh vegetables, prompting a growing public health concern. Most studies regarding interactions of STEC on fresh produce focused on E. coli O157:H7. Limited information is available about survival or fitness of E. coli O104:H4, non-O157 pathogen that was linked to one of the largest outbreaks of hemolytic uremic syndrome in 2011. In this study, survival of E. coli O104:H4 was evaluated on Arabidopsis thaliana plant and lettuce for 5 days compared with E. coli O157:H7, and expression of pathogenesis-realted gene (PR1; induction of plant defense response) was examined by reverse transcription quantitative PCR, and potential influence of capsular polysaccharide (CPS) on the bacterial fitness on plant was investigated. Populations of E. coli O104:H4 strains (RG1, C3493, and LpfA) on Arabidopsis and lettuce were significantly (P < 0.05) greater than those of E. coli O157:H7 strains (7386 and sakai) at day 5 post-inoculation, indicating E. coli O104:H4 may have better survival ability on the plants. In addition, the E. coli O104:H4 strains produced significantly (P < 0.05) higher amounts of CPS compared with the E. coli O157:H7 strains. RG1 strain (1.5-fold) initiated significantly (P < 0.05) lower expression of PR1 gene indicating induction of plant defense response compared with E. coli O157:H7 strains 7386 (2.9-fold) and sakai (2.7-fold). Collectively, the results in this study suggests that different level of CPS production and plant defense response initiated by each STEC strain might influence the bacterial survival or persistence on plants. The present study provides better understanding of survival behavior of STEC, particularly E. coli O104:H4, using a model plant and vegetable under pre-harvest conditions with plant defense response.

In silico prediction of active site and in vitro DNase and RNase activities of Helicoverpa-inducible pathogenesis related-4 protein from Cicer arietinum

Plants are endowed with an innate immune system, which enables them to protect themselves from pest and pathogen. The participation of pathogenesis-related (PR) proteins is one of the most crucial events of inducible plant defense response. Herein, we report the characterization of CaHaPR-4, a Helicoverpa-inducible class II PR-4 protein from chickpea. Bioinformatic analysis of CaHaPR-4 protein indicated the presence of a signal peptide, barwin domain but it lacks the chitin-binding site/hevein domain. The recombinant CaHaPR-4 is bestowed with RNase and bivalent ion-dependent DNase activity. Further, the RNA and DNA binding sites were identified and confirmed by analyzing interactions between mutated CaHaPR-4 with the altered active site and ribonuclease inhibitor, 5′ADP and DNase inhibitor, 2‑nitro‑5‑thiocyanobenzoic acid (NTCB) using 3D modeling and docking studies. Moreover, CaHaPR-4 shows antifungal activity as well as growth inhibiting properties against neonatal podborer larvae. To the best of our knowledge, this is the first report of a PR-4 showing RNase, DNase, antifungal and most importantly insect growth inhibiting properties against Helicoverpa armigera simultaneously.

Consequences of Sphaeropsis tip blight disease for the phytohormone profile and antioxidative metabolism of its pine host.

Phytopathogenic fungi infections induce plant defence responses that mediate changes in metabolic and signalling processes with severe consequences for plant growth and development. Sphaeropsis tip blight, induced by the endophytic fungus Sphaeropsis sapinea that spreads from stem tissues to the needles, is the most widespread disease of conifer forests causing dramatic economic losses. However, metabolic consequences of this disease on bark and wood tissues of its host are largely unexplored. Here, we show that diseased host pines experience tissue dehydration in both bark and wood. Increased cytokinin and declined indole-3-acetic acid levels were observed in both tissues and increased jasmonic acid and abscisic acid levels exclusively in the wood. Increased lignin contents at the expense of holo-cellulose with declined structural biomass of the wood reflect cell wall fortification by S.sapinea infection. These changes are consistent with H2 O2 accumulation in the wood, required for lignin polymerization. Accumulation of H2 O2 was associated with more oxidized redox states of glutathione and ascorbate pools. These findings indicate that S.sapinea affects both phytohormone signalling and the antioxidative defence system in stem tissues of its pine host during the infection process.

Corrigendum: Disruption of OsSEC3A increases the content of salicylic acid and induces plant defense responses in rice.

Corrigendum: Disruption of OsSEC3A increases the content of salicylic acid and induces plant defense responses in rice.

The sesquiterpene botrydial from Botrytis cinerea induces phosphatidic acid production in tomato cell suspensions.

The phytotoxin botrydial triggers PA production in tomato cell suspensions via PLD and PLC/DGK activation. PLC/DGK-derived PA is partially required for botrydial-induced ROS generation. Phosphatidic acid (PA) is a phospholipid second messenger involved in the induction of plant defense responses. It is generated via two distinct enzymatic pathways, either via phospholipase D (PLD) or by the sequential action of phospholipase C and diacylglycerol kinase (PLC/DGK). Botrydial is a phytotoxic sesquiterpene generated by the necrotrophic fungus Botrytis cinerea that induces diverse plant defense responses, such as the production of reactive oxygen species (ROS). Here, we analyzed PA and ROS production and their interplay upon botrydial treatments, employing tomato (Solanum lycopersicum) cell suspensions as a model system. Botrydial induces PA production within minutes via PLD and PLC/DGK. Either inhibition of PLC or DGK diminishes ROS generation triggered by botrydial. This indicates that PLC/DGK is upstream of ROS production. In tomato, PLC is encoded by a multigene family constituted by SlPLC1-SlPLC6 and the pseudogene SlPLC7. We have shown that SlPLC2-silenced plants have reduced susceptibility to B. cinerea. In this work, we studied the role of SlPLC2 on botrydial-induced PA production by silencing the expression of SlPLC2 via a specific artificial microRNA. Upon botrydial treatments, SlPLC2-silenced-cell suspensions produce PA levels similar to wild-type cells. It can be concluded that PA is a novel component of the plant responses triggered by botrydial.

Comparative assessment of insect pests population densities of three selected cucurbit crops

Comparative assessment of insect pests population densities of three selected cucurbit crops

Disruption of OsSEC3A increases the content of salicylic acid and induces plant defense responses in rice.

The exocyst, an evolutionarily conserved octameric protein complex involved in exocytosis, has been reported to be involved in diverse aspects of morphogenesis in Arabidopsis. However, the molecular functions of such exocytotic molecules in rice are poorly understood. Here, we examined the molecular function of OsSEC3A, an important subunit of the exocyst complex in rice. The OsSEC3A gene is expressed in various organs, and OsSEC3A has the potential ability to participate in the exocyst complex by interacting with several other exocyst subunits. Disruption of OsSEC3A by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) caused dwarf stature and a lesion-mimic phenotype. The Ossec3a mutant exhibited enhanced defense responses, as shown by up-regulated transcript levels of pathogenesis- and salicylic acid synthesis-related genes, increased levels of salicylic acid, and enhanced resistance to the fungal pathogen Magnaporthe oryzae. Subcellular localization analysis demonstrated that OsSEC3A has a punctate distribution with the plasma membrane. In addition, OsSEC3A interacted with rice SNAP25-type t-SNARE protein OsSNAP32, which is involved in rice blast resistance, via the C-terminus and bound to phosphatidylinositol lipids, particularly phosphatidylinositol-3-phosphate, through its N-terminus. These findings uncover the novel function of rice exocyst subunit SEC3 in defense responses.

A Verticillium dahliae Extracellular Cutinase Modulates Plant Immune Responses.

Cutinases have been implicated as important enzymes during the process of fungal infection of aerial plant organs. The function of cutinases in the disease cycle of fungal pathogens that invade plants through the roots has been less studied. Here, functional analysis of 13 cutinase (carbohydrate esterase family 5 domain-containing) genes (VdCUTs) in the highly virulent vascular wilt pathogen Verticillium dahliae Vd991 was performed. Significant sequence divergence in cutinase family members was observed in the genome of V. dahliae Vd991. Functional analyses demonstrated that only VdCUT11, as purified protein, induced cell death and triggered defense responses in Nicotiana benthamiana, cotton, and tomato plants. Virus-induced gene silencing showed that VdCUT11 induces plant defense responses in Nicotiana benthamania in a BAK1 and SOBIR-dependent manner. Furthermore, coinfiltration assays revealed that the carbohydrate-binding module family 1 protein (VdCBM1) suppressed VdCUT11-induced cell death and other defense responses in N. benthamiana. Targeted deletion of VdCUT11 in V. dahliae significantly compromised virulence on cotton plants. The cutinase VdCUT11 is an important secreted enzyme and virulence factor that elicits plant defense responses in the absence of VdCBM1.

Extracellular ATP Acts on Jasmonate Signaling to Reinforce Plant Defense.

Damaged cells send various signals to stimulate defense responses. Recent identification and genetic studies of the plant purinoceptor, P2K1 (also known as DORN1), have demonstrated that extracellular ATP is a signal involved in plant stress responses, including wounding, perhaps to evoke plant defense. However, it remains largely unknown how extracellular ATP induces plant defense responses. Here, we demonstrate that extracellular ATP induces plant defense mediated through activation of the intracellular signaling of jasmonate (JA), a well-characterized defense hormone. In Arabidopsis (Arabidopsis thaliana) leaves, ATP pretreatment induced resistance against the necrotrophic fungus, Botrytis cinerea The induced resistance was enhanced in the P2K1 receptor overexpression line, but reduced in the receptor mutant, dorn1-3 Mining the transcriptome data revealed that ATP induces a set of JA-induced genes. In addition, the P2K1-associated coexpression network contains defense-related genes, including those encoding jasmonate ZIM-domain (JAZ) proteins, which play key roles as repressors of JA signaling. We examined whether extracellular ATP impacts the stability of JAZ1 in Arabidopsis. The results showed that the JAZ1 stability decreased in response to ATP addition in a proteasome-dependent manner. This reduction required intracellular signaling via second messengers-cytosolic calcium, reactive oxygen species, and nitric oxide. Interestingly, the ATP-induced JAZ1 degradation was attenuated in the JA receptor mutant, coi1, but not in the JA biosynthesis mutant, aos, or upon addition of JA biosynthesis inhibitors. Immunoprecipitation analysis demonstrated that ATP increases the interaction between COI1 and JAZ1, suggesting direct cross talk between extracellular ATP and JA in intracellular signaling events. Taken together, these results suggest that extracellular ATP signaling directly impacts the JA signaling pathway to maximize plant defense responses.

CONTROL OF FIRE BLIGHT INFECTIONS WITH SYNTHETIC PEPTIDES THAT ELICIT PLANT DEFENSE RESPONSES

Antimicrobial peptides (AMPs) are reliable compounds to develop strategies for integrated plant disease management. These peptides can act directly against the pathogens and/or induce plant defense responses. In this work, 33 sequences including linear and cyclic peptides, linear and cyclic peptidotriazoles, and cyclic lipopeptides were tested for induction of plant defense. A first screening was carried out with BY2 tobacco cell cultures by determining the alkalinization and production of hydrogen peroxide. Peptides BP13, BP100, BP143 and BPC200W were slightly positive in these assays and were tested by RT-qPCR for expression of 10 defense-related genes in tomato, which are representative of the jasmonic acid, salicylic acid and ethylene pathways. BP13 and BPC200W induced overexpression of several genes that are also overexpressed by reference plant defense peptide elicitors. Plant assays with potted pear plants in the greenhouse confirmed the effect of peptides BP13 and BPC200W in controlling Erwinia amylovora infections, putatively through plant defense induction.

Silicon amendment is involved in the induction of plant defense responses to a phloem feeder

Plant resistance to herbivores is a key component in integrated pest management. In most cases, silicon (Si) amendment to plants enhances resistance to herbivorous insects. The increase of plant physical barrier and altered insect behaviors are proposed as mechanisms for the enhanced resistance in Si-amended plants, but our understanding of the induced mechanisms involved in Si-enhanced plant resistance to phloem-feeding insects remains unclear. Here, we show that Si amendment to rice (Oryza sativa) plants impacts multiple plant defense responses induced by a phloem-feeder, the brown planthopper (Nilaparvata lugens, BPH). Si amendment improved silicification of leaf sheaths that BPH feed on. Si addition suppressed the increase of malondialdehyde concentration while encouraged increase of H2O2 concentration in plants attacked by BPH. Higher activities of catalase and superoxide dismutase were recorded in Si-amended than in non-amended BPH-infested plants. BPH infestation activated synthases for secondary metabolites, polyphenol oxidase and pheny-lalanine ammonia-lyase, and β-1,3-glucanase, but the activation was greater in Si-amended than in non-amended plants. Taken together, our findings demonstrate that Si amendment interacts with BPH infestation in the induction of plant defense responses and consequently, to confer enhanced rice plant resistance.

Involvement of Trichoderma harzianum Epl-1 Protein in the Regulation of Botrytis Virulence- and Tomato Defense-Related Genes.

Several Trichoderma spp. are well known for their ability to: (i) act as important biocontrol agents against phytopathogenic fungi; (ii) function as biofertilizers; (iii) increase the tolerance of plants to biotic and abiotic stresses; and (iv) induce plant defense responses via the production and secretion of elicitor molecules. In this study, we analyzed the gene-regulation effects of Trichoderma harzianum Epl-1 protein during the interactions of mutant Δepl-1 or wild-type T. harzianum strains with: (a) the phytopathogen Botrytis cinerea and (b) with tomato plants, on short (24 h hydroponic cultures) and long periods (4-weeks old plants) after Trichoderma inoculation. Our results indicate that T. harzianum Epl-1 protein affects the in vitro expression of B. cinerea virulence genes, especially those involved in the botrydial biosynthesis (BcBOT genes), during the mycoparasitism interaction. The tomato defense-related genes were also affected, indicating that Epl-1 is involved in the elicitation of the salicylic acid pathway. Moreover, Epl-1 also regulates the priming effect in host tomato plants and contributes to enhance the interaction with the host tomato plant during the early stage of root colonization.

Bacterial and plant natriuretic peptides improve plant defence responses against pathogens.

Plant natriuretic peptides (PNPs) have been implicated in the regulation of ions and water homeostasis, and their participation in the plant immune response has also been proposed. Xanthomonas citri ssp. citri contains a gene encoding a PNP-like protein (XacPNP) which has no homologues in other bacteria. XacPNP mimics its Arabidopsis thaliana homologue AtPNP-A by modifying host responses to create favourable conditions for pathogen survival. However, the ability of XacPNP to induce plant defence responses has not been investigated. In order to study further the role of XacPNP in vivo, A. thaliana lines over-expressing XacPNP, lines over-expressing AtPNP-A and AtPNP-A-deficient plants were generated. Plants over-expressing XacPNP or AtPNP-A showed larger stomatal aperture and were more resistant to saline or oxidative stress than were PNP-deficient lines. In order to study further the role of PNP in biotic stress responses, A. thaliana leaves were infiltrated with pure recombinant XacPNP, and showed enhanced expression of genes related to the defence response and a higher resistance to pathogen infections. Moreover, AtPNP-A expression increased in A. thaliana on Pseudomonas syringae pv. tomato (Pst) infection. This evidence led us to analyse the responses of the transgenic plants to pathogens. Plants over-expressing XacPNP or AtPNP-A were more resistant to Pst infection than control plants, whereas PNP-deficient plants were more susceptible and showed a stronger hypersensitive response when challenged with non-host bacteria. Therefore, XacPNP, acquired by horizontal gene transfer, is able to mimic PNP functions, even with an increase in plant defence responses.

Alpha-momorcharin enhances Tobacco mosaic virus resistance in tobaccoNN by manipulating jasmonic acid-salicylic acid crosstalk

Alpha-momorcharin (α-MMC) is a type-I ribosome inactivating protein (RIP) with a molecular weight of 29 kDa found in plants. This protein has been shown to be effective against a broad range of human viruses and also has anti-tumor activities. However, the mechanism by which α-MMC induces plant defense responses and regulates the N gene to promote resistance to the Tobacco mosaic virus (TMV) is still not clear. By using pharmacological and infection experiments, we found that α-MMC enhances TMV resistance of tobacco plants containing the N gene (tobaccoNN). Our results showed that plants pretreated with 0.5 mg/ml α-MMC could relieve TMV-induced oxidative damage, had enhanced the expression of the N gene and increased biosynthesis of jasmonic acid (JA) and salicylic acid (SA). Moreover, transcription of JA and SA signaling pathway genes were increased, and their expression persisted for a longer period of time in plants pretreated with α-MMC compared with those pretreated with water. Importantly, exogenous application of 1-Aminobenzotriazole (ABT, SA inhibitor) and ibuprofen (JA inhibitor) reduced α-MMC induced plant resistance under viral infection. Thus, our results revealed that α-MMC enhances TMV resistance of tobaccoNN plants by manipulating JA-SA crosstalk.

Tarımsal Üretimde Yararlanılan Trichoderma Ürünleri ve Metabolitleri

Phytopathogenic agents cause considerable crop losses incurred by plant diseases worldwide. The most goverments in World, have started to implement legislative mandates in order to reduce the pesticide applications and dependency in agriculture and increase the environmental and consumers safety. By implementing integrated pest management [IPM], it is aimed to establish the necessary conditions to employ these practices. Trichoderma is among the best known and widely used fungal genera as biocontrol agent. Products of Trichoderma and itself, are promoted as bio-pesticide, bio-fungicide, bio-inoculant, bio-stimulant, bio-decomposer, bio-fertilizer, plant growth stimulator. Main mechanisms underlying its beneficial properties are; mycoparasitism, hyperparasitism, antibiosis, competition, cell wall lytic enzymes, enhancement of plant growth, acquisiton of soil nutrients, induction of plant defense responses. In this review, we focused on the Trichoderma and its secondary metabolites that are used in agriculture and their interactions between phytopathogens and plants.