Redox Status, JA and ET Signaling Pathway Regulating Responses to Botrytis cinerea Infection Between the Resistant Cucumber Genotype and Its Susceptible Mutant

Abstract

Botrytis cinerea is an important necrotrophic fungal pathogen with a broad host range and the ability to causing great economic losses in cucumber. However, the resistance mechanism against this pathogen in cucumber was not well understood. In this study, the microscopic observation of the spore growth, redox status measurements and transcriptome analysis were carried out after Botrytis cinerea infection in the resistant genotype No.26 and its susceptible mutant 26M. Results revealed shorter hypha, lower rate of spore germination, less acceleration of H₂O₂, O₂ ^-, and lower total glutathione content (GSH+GSSG) in No.26 than that in 26M, which were identified by the staining result of DAB and NBT. Transcriptome data showed that after pathogen infection, a total of 3901 and 789 different expression genes (DEGs) were identified in No.26 and 26M respectively. These DEGs were highly enriched in redox regulation pathway, hormone signaling pathway and plant-pathogen interaction pathway. The glutathione S-transferase genes, putative peroxidase gene, and NADPH oxidase were up-regulated in No.26 whereas these genes changed little in 26M after Botrytis cinerea infection. Jasmonic acid and ethylene biosynthesis and signaling pathways were distinctively activated in No.26 comparing with 26M upon infection. Much more plant defense related genes including mitogen-activated protein kinases, calmodulin, calmodulin-like protein, calcium-dependent protein kinase, and WRKY transcription factor were induced in No.26 than 26M after pathogen infection. Finally, a model was established which elucidated the resistance difference between resistant cucumber genotype and susceptible mutant after B. cinerea infection.

Keywords: B. cinerea; cucumber; redox status; resistant; signaling pathway.

Figure 1

Symptom development in susceptible (26M) and resistant (No. 26) cucumber. (A) Disease symptoms observed at 0, 2, 3, 4, 5, 6 days post-inoculation (dpi). (B) The lesion area at 3, 4, 5, 6 days post-inoculation (dpi) in susceptible and resistant cucumber; (C) The length of primary hyphal at 2 days post-inoculation (dpi) in susceptible and resistant cucumber; (D) The germination of spores at 1 day post-inoculation (dpi) in susceptible and resistant cucumber. (E) Growth of B. cinerea was observed at 1, 2, 3, 5 days post-inoculation (dpi). Red arrow: conidium; Yellow arrow: stomata; Black arrow: germ tube; Green arrow: callose; hy: hypha; Fifteen plants per cucumber genotype were infected with B. cinerea and lesion sizes were measured. The means of lesion size data from five independent experiments are shown (mean ± SD). Five seedlings per replicate and three replicates are included for staining. Asterisk indicated the statistically significant difference between different cucumber genotypes after B. cinerea infection (p < 0.05). Bar=50 μm.

Figure 2

Content of hydrogen peroxide, superoxide anion. (A) DAB and NBT staining; (B1) the content of H₂O₂; (B2) the content of O₂ ^-; (B3) the content of total glutathione and (B4) the content of oxidized glutathione. At least five seedlings per replicate and three replicates are included for determining content of H₂O₂ and O₂ ^-. Asterisk indicated the statistically significant difference between different cucumber genotypes after B. cinerea infection (p < 0.05). Bar=50 μm (at 4 hpi). Bar=200 μm (at 12 hpi, 16 hpi, 20 hpi).

Figure 3

Expression patterns of unigenes differentially expressed after leaf inoculation with B. cinerea. (A) Experimental design; (B) The number of differentially expressed genes after inoculation with B. cinerea at 2 days post-inoculation (dpi) in the susceptible and resistant cucumber; (C) The number of up and down regulated expression genes after inoculation with B. cinerea at 2 days post-inoculation (dpi) in the susceptible and resistant cucumber. (D, E) The analysis of metabolite pathway in resistant and susceptible cucumber under B. cinerea infection.

Figure 4

Differentially expressed genes related to ROS regulation at 2 days post-inoculation (dpi) in the susceptible and resistant cucumber (B) and some key substances involved in ROS regulation (A). RBOH: respiratory burst oxidase homologue; GST: glutathione S-transferase; GRX: glutaredoxin; AsA: ascorbate and DHA: dehydroascorbate. Red box represented up-regulation of genes or increment in content.

Figure 5

Differentially expressed genes involved in hormone signal pathway at 2 days post-inoculation (dpi) in the susceptible and resistant cucumber. DEGs in ET signal (A) and JA signal (B). (C) Simplified ET signal transduction induced by B. cinerea infection. (D) Simplified JA signal transduction induced by B. cinerea infection. 13-HPOT (13(S)-9(Z), ll(E), 15(Z)-Hydroperoxyoctade-catrienoic acid); LOX (Lipoxygenase); JA (jasmonate); JAR1 (jasmonate resistant 1); SCF (Skp/Cullin/F-box); COI1 (coronatine-insensitive 1); JA-Ile (jasmonate-isoleucine); JAZ (jasmonate ZIM domain protein); MYC (MYC transcription factor); SAM (S′ adenosyl-l-methionine); ACS (1-aminocyclopropane-1-carboxylic acid synthase); ACC (1-aminocyclopropane-1-carboxylic acid); ACO (1- aminocyclopropane-1-carboxylate oxidase); ET (ethylene); ETR1 (ethylene-response gene 1); CTR1 (constitutive triple response); EIN3 (ethylene insensitive 3); ERF (ethylene-responsive transcription factor). Red box represented up-regulation of genes.

Figure 6

Differentially expressed genes involved in PAL pathway at 2 days post-inoculation (dpi) in the susceptible and resistant cucumber (B) and simplified PAL pathway induced by B. cinerea infection (A) PAL (phenylalanine ammonia-lyase); CCR (cinnamoyl-CoA reductase); CAD (cinnamoyl alcohol dehydrogenase); POX (peroxidase). Red box represented up-regulation of genes.

Figure 7

Differentially expressed genes involved in basal defense at 2 days post-inoculation (dpi) in the susceptible and resistant cucumber (B) and simplified basal defense induced by B. cinerea infection (A). MAPK (mitogen-activated protein kinase); CaM (calmodulin), CaML (calmodulin-like protein); CaMK (calcium-dependent protein kinase).

Figure 8

Cellular model summarizing potential mechanism of cucumber resistance to B. cinerea. Red represented the higher level of gene expression and much accumulation of content; Green represented the less accumulation of content. Orange represented the lower level of gene expression.