Chitosan primes plant defence mechanisms against Botrytis cinerea including Avr9/Cf-9 rapidly-elicited genes
ABSTRACT: Current protection strategies against the fungal pathogen Botrytis cinerea rely on a combination of conventional fungicides and host genetic resistance. Defence elicitors can stimulate plant defence mechanisms through a phenomenon known as priming. Priming results on a faster and/or stronger expression of resistance upon pathogen attack. This work aims to study priming of a commercial formulation of the elicitor Chitosan. Treatments with Chitosan result in induced resistance in solanaceous and brassicaceous plants. Large-scale transcriptomic analysis in this study revealed that Chitosan primes gene expression at early time-points after infection. Four conditions were analysed using microarrays: (i) water-treated and non-infected plants (Water + Mock); (ii) Chitosan-treated and non-infected plants (Chitosan + Mock); (iii) water-treated and B. cinerea-infected plants (Water + B. cinerea); (iv) Chitosan-treated and B. cinerea-infected plants (Chitosan + B. cinerea). Inoculations were performed four days after treatment with Chitosan, and leaf discs from four independent plants (biological replicates) per treatment were sampled at 6 h, 9 h and 12 h post-inoculation (hpi) with water mock or B. cinerea spores.
Project description:Microbe-associated molecular pattern (MAMP)-triggered immunity (MTI) is the first layer of molecular defense encountered by pathogens when they attempt to infect plants. MTI is dependent on cell surface pattern-recognition receptors (PRR) which act upstream of mitogen-activated protein kinase (MAPK) pathways. Genetic screening and mutant knock-out lines have largely contributed to our knowledge of MTI. However, genetic screening is confined to phenotype-causing mutations and scarcely enables the discovery of redundantly-acting proteins. We sought to discover protein components that contribute in MTI, using a phenotype-independent approach to discern nucleus-localized proteins after MTI induction. We report on the nuclear proteome of Columbia-0 (Col-0) and chitin elicitor receptor kinase 1 (cerk1) mutant plants 15 min after MTI induction. Our approach revealed that MAMP-treated cerk1 plants had many proteins in common with Col-0-treated plants following chitosan treatment. cerk1 plants also manifested several unique proteins that were absent from Col-0 plants when elicited with chitosan indicating that they also perceive chitosan. Detailed analysis of the identified proteins revealed a nuclear accumulation of transcriptional regulators and transcription factors, DNA-modifying enzymes, RNA-binding proteins and ribosomal proteins. No novel MAPKs were found although Receptor for activated C kinase 1, a scaffold protein involved in defense, and a nucleotide binding leucine-rich repeat protein, implicated in resistance to Leptosphaeria maculans, were discovered in the nucleus of chitosan-treated plants and absent from water-treated control plants.
Project description: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.
Project description:The Chilean strawberry (Fragaria chiloensis) fruit has interesting organoleptic properties, but its postharvest life is affected by gray mold decay caused by Botrytis cinerea. The effect of preharvest applications of methyl jasmonate (MeJA) or chitosan on the molecular defense-related responses and protection against gray mold decay were investigated in Chilean strawberry fruit during postharvest storage. Specifically, we inoculated harvested fruit with B. cinerea spores and studied the expression of genes encoding for the pathogenesis-related (PR) proteins ?-1,3-glucanases (FcBG2-1, FcBG2-2 and FcBG2-3) and chitinases (FcCHI2-2 and FcCHI3-1), and for polygalacturonase inhibiting proteins (FcPGIP1 and FcPGIP2) at 0, 2, 24, 48, and 72 h post inoculation (hpi). Remarkably, MeJA- and chitosan-treated fruit exhibited a lower incidence of B. cinerea infection than the control-treated at 48 and 72 hpi. At the molecular level, both are efficient elicitors for priming in F. chiloensis fruit since we observed an upregulation of the FcBG2-1, FcBG2-3, FcPGIP1, and FcPGIP2 at 0 hpi. Moreover, a chitosan-mediated upregulation of FcPGIPs at early times post inoculation (2-24 hpi) and MeJA upregulated FcBGs (24-72 hpi) and FcPGIP1 at later times could contribute to reduce B. cinerea incidence by differential upregulation of defense genes. We concluded that preharvest applications of MeJA or chitosan had a long-lasting effect on the reduction of B. cinerea incidence during postharvest as well as an enhancer effect on the induction of PR and PGIP gene expression.
Project description:We treated Arabidopsis seedlings with chitosan and carried out a transcript profiling analysis (GeneChip microarrays) in order to identify genes and transcription factors regulated by chitosan. The results showed that jasmonate and defense responsive genes, camalexin and lignin biosynthetic genes were among genes up-regulated by chitosan. Several transcription factors are also strongly induced by chitosan. The results suggested that chitosan can be used as a strong elicitor of defense pathways. Experiment Overall Design: Arabidopsis thaliana, ecotype Columbia (Col-0) seeds were sterilized for 7 min in 1.7% (v/v) bleach solution, incubated over night in 4% PPM (Plant Preservative Mixture, Plant Cell Technology, Washington DC, USA) in a full strength sterilized Murashige-Skoog (MS) salt solution with gentle shaking. Subsequently, the seeds were rinsed in abundant sterile water and transferred into 2.5 ml liquid growing media (MS half strength solution) with 0.05% PPM in 6-well plates. The plates were incubated in the dark at 4 °C for two days and finally transferred to continuous light (90µm photons/ m-2) with gentle swirling for four days in a plant growth chamber at 22 °C. Experiment Overall Design: . Treatments were performed by comparing a control solution containing the solvent used to solubilize chitosan, and a chitosan solution concentrated 150 µg/ml.
Project description:Fungal leaf diseases cause economically important damage to crop plants. Protective treatments help producers to secure good quality crops. In contrast, curative treatments based on visually detectable symptoms are often riskier and less effective because diseased crop plants may develop disease symptoms too late for curative treatments. Therefore, early disease detection prior symptom development would allow an earlier, and therefore more effective, curative management of fungal diseases. Using a five-lens multispectral imager, spectral reflectance of green, blue, red, near infrared (NIR, 840 nm), and rededge (RE, 720 nm) was recorded in time-course experiments of detached tomato leaves inoculated with the fungus Botrytis cinerea and mock infection solution. Linear regression models demonstrate NIR and RE as the two most informative spectral data sets to differentiate pathogen- and mock-inoculated leaf regions of interest (ROI). Under controlled laboratory conditions, bands collecting NIR and RE irradiance showed a lower reflectance intensity of infected tomato leaf tissue when compared with mock-inoculated leaves. Blue and red channels collected higher intensity values in pathogen- than in mock-inoculated ROIs. The reflectance intensities of the green band were not distinguishable between pathogen- and mock infected ROIs. Predictions of linear regressions indicated that gray mold leaf infections could be identified at the earliest at 9 h post infection (hpi) in the most informative bands NIR and RE. Re-analysis of the imagery taken with NIR and RE band allowed to classify infected tissue.
Project description:Sound vibration (SV), a mechanical stimulus, can trigger various molecular and physiological changes in plants like gene expression, hormonal modulation, induced antioxidant activity and calcium spiking. It also alters the seed germination and growth of plants. In this study, we investigated the effects of SV on the resistance of Arabidopsis thaliana against Botrytis cinerea infection. The microarray analysis was performed on infected Arabidopsis plants pre-exposed to SV of 1000 Hertz with 100 decibels. Broadly, the transcriptomic analysis revealed up-regulation of several defense and SA-responsive and/or signaling genes. Quantitative real-time PCR (qRT-PCR) analysis of selected genes also validated the induction of SA-mediated response in the infected Arabidopsis plants pre-exposed to SV. Corroboratively, hormonal analysis identified the increased concentration of salicylic acid (SA) in the SV-treated plants after pathogen inoculation. In contrast, jasmonic acid (JA) level in the SV-treated plants remained stable but lower than control plants during the infection. Based on these findings, we propose that SV treatment invigorates the plant defense system by regulating the SA-mediated priming effect, consequently promoting the SV-induced resistance in Arabidopsis against B. cinerea.
Project description:Mycorrhizal plants display enhanced resistance to several pathogens. However, the molecular mechanisms regulating mycorrhiza-induced resistance (MIR) are still elusive. We aim to study the mechanisms underlying MIR against Botrytis cinerea and the role of callose accumulation during this process. Mycorrhizal tomato plants inoculated with Rhizoglomus irregularis displayed callose priming upon B. cinerea infection. The callose inhibitor 2-deoxy-d-glucose abolished MIR, confirming the relevance of callose in the bioprotection phenomena. While studying the mechanisms underlying mycorrhiza-induced callose priming, we found that mycorrhizal plants display an enhanced starch degradation rate that is correlated with increased levels of ?-amylase1 transcripts following pathogen infection. Starch mobilization in mycorrhizal plants seems coordinated with the increased transcription of sugar transporter and invertase genes. Moreover, the expression levels of genes encoding the vesicular trafficking proteins ATL31 and SYP121 and callose synthase PMR4 were higher in the mycorrhizal plants and further boosted by subsequent pathogen infection. All these proteins play a key role in the priming of callose accumulation in Arabidopsis, suggesting that callose priming is an induced resistance mechanism conserved in different plant species. This evidence highlights the importance of sugar mobilization and vesicular trafficking in the priming of callose as a defence mechanism in mycorrhiza-induced resistance.
Project description:In compatible interactions, biotrophic microbial phytopathogens rely on the supply of assimilates by the colonized host tissue. It has been found in rice that phloem localized SWEET sucrose transporters can be reprogrammed by bacterial effectors to establish compatibility. We observed that sweet11/sweet12 double mutants, but not single mutants, exhibited increased resistance toward the fungal hemibiotroph Colletotrichum higginsianum (Ch), both in the biotrophic and the necrotrophic colonization phase. We therefore investigated if the phloem localized transporters AtSWEET11 and AtSWEET12 represent additive susceptibility factors in the interaction of Arabidopsis with Ch. AtSWEET12-YFP fusion protein driven by the endogenous promoter strongly accumulated at Ch infection sites and in the vasculature upon challenge with Ch. However, susceptibility of sweet12 single mutants to Ch was comparable to wild type, indicating that the accumulation of AtSWEET12 at Ch infection sites does not play a major role for compatibility. AtSWEET12-YFP reporter protein was not detectable at the plant-pathogen interface, suggesting that AtSWEET12 is not targeted by Ch effectors. AtSWEET11-YFP accumulation in pAtSWEET11:AtSWEET11-YFP plants were similar in Ch infected and mock control leaves. A close inspection of major carbohydrate metabolism in non-infected control plants revealed that soluble sugar and starch content were substantially elevated in sweet11/sweet12 double mutants during the entire diurnal cycle, that diurnal soluble sugar turnover was increased more than twofold in sweet11/sweet12, and that accumulation of free hexoses and sucrose was strongly expedited in double mutant leaves compared to wild type and both single mutants during the course of Ch infection. After 2 days of treatment, free and conjugated SA levels were significantly increased in infected and mock control leaves of sweet11/sweet12 relative to all other genotypes, respectively. Induced genes in mock treated sweet11/sweet12 leaves were highly significantly enriched for several GO terms associated with SA signaling and response compared to mock treated wild-type leaves, indicating sugar-mediated priming of the SA pathway in the double mutant. Infection assays with salicylic acid deficient sweet11/sweet12/sid2 triple mutants demonstrated that reduced susceptibility observed in sweet11/sweet12 was entirely dependent on the SA pathway. We suggest a model how defects in phloem loading of sucrose can influence SA priming and hence, compatibility.
Project description:Plants harbor various beneficial bacteria that modulate their innate immunity, resulting in induced systemic resistance (ISR) against various pathogens. However, the immune mechanisms underlying ISR triggered by Bacillus spp. and Pseudomonas spp. against pathogens with different lifestyles are not yet clearly elucidated. Here, we show that root drenching of Arabidopsis plants with Pseudomonas fluorescensPTA-CT2 and Bacillus subtilis PTA-271 can induce ISR against the necrotrophic fungus B. cinerea and the hemibiotrophic bacterium Pseudomonas syringae Pst DC3000. In the absence of pathogen infection, both beneficial bacteria do not induce any consistent change in systemic immune responses. However, ISR relies on priming faster and robust expression of marker genes for the salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) signaling pathways upon pathogen challenge. These responses are also associated with increased levels of SA, JA, and abscisic acid (ABA) in the leaves of bacterized plants after infection. The functional study also points at priming of the JA/ET and NPR1-dependent defenses as prioritized immune pathways in ISR induced by both beneficial bacteria against B. cinerea. However, B. subtilis-triggered ISR against Pst DC3000 is dependent on SA, JA/ET, and NPR1 pathways, whereas P. fluorescens-induced ISR requires JA/ET and NPR1 signaling pathways. The use of ABA-insensitive mutants also pointed out the crucial role of ABA signaling, but not ABA concentration, along with JA/ET signaling in primed systemic immunity by beneficial bacteria against Pst DC3000, but not against B. cinerea. These results clearly indicate that ISR is linked to priming plants for enhanced common and distinct immune pathways depending on the beneficial strain and the pathogen lifestyle.
Project description:Comparison of chitosan-treated B. cereus ATCC 14579 cells with non-treated B. cereus ATCC 14579 cells. 2 chitosans with similar molecular weight (Mw) but different degrees of acetylation (Fa) were used: chitosan B (Mw: 28.4 kDa, Fa: 0.16) (samples 1-3) and chitosan A (Mw: 36.0 kDa, Fa: 0.01) (samples 4-6). One-condition design comparision of treated vs. non-treated control. 3 biological replicates, including a dye swap.