GhERF-IIb3 regulates the accumulation of jasmonate and leads to enhanced cotton resistance to blight disease

Abstract

The phytohormone jasmonic acid (JA) and its derivatives, collectively referred to as jasmonates, regulate many developmental processes, but are also involved in the response to numerous abiotic/biotic stresses. Thus far, powerful reverse genetic strategies employing perception, signalling or biosynthesis mutants have broadly contributed to our understanding of the role of JA in the plant stress response and development, as has the chemical gain-of-function approach based on exogenous application of the hormone. However, there is currently no method that allows for tightly controlled JA production in planta. By investigating the control of the JA synthesis pathway in bacteria-infected cotton (Gossypium hirsutum L.) plants, we identified a transcription factor (TF), named GhERF-IIb3, which acts as a positive regulator of the JA pathway. Expression of this well-conserved TF in cotton leaves was sufficient to produce in situ JA accumulation at physiological concentrations associated with an enhanced cotton defence response to bacterial infection.

Keywords: ERF transcription factor; cotton; defence response; jasmonate, Xanthomonas citri pv. malvacearum.

Figure 1

Wounding promotes both the up‐regulation of jasmonic acid (JA) biosynthesis‐related gene expression and oxo‐phytodienoic acid (OPDA)/JA accumulation in cotton cotyledons. (A) Twelve‐day‐old cotton plants were wounded four times with a haemostat. Damaged cotyledons were harvested at the indicated time points after wounding. OPDA/JA levels were determined by liquid chromatography‐mass spectrometry (LC‐MS). (B) Expression of cotton ALLENE OXIDE SYNTHASE‐like (GhAOS‐like), ALLENE OXIDE CYCLASE 2‐like (GhAOC2‐like) and ACYL‐COENZYME A OXIDASE‐like 1a (GhACX1a‐like) genes in damaged cotyledons was quantified by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Results in (A) and (B) are averages of three biological replicates; error bars indicate standard deviation (n > 12).

Figure 2

Coordinated induction of jasmonic acid (JA) biosynthesis‐related gene expression and metabolite accumulation on challenge with the avirulent strain of Xanthomonas citri pv. malvacearum (Xcm). (A) Time course expression of JA biosynthesis‐related genes cotton ALLENE OXIDE SYNTHASE‐like (GhAOS‐like), ALLENE OXIDE CYCLASE 2‐like (GhAOC2‐like) and ACYL‐COENZYME A OXIDASE‐like 1a (GhACX1a‐like) in response to Xanthomonas virulent (Xcm20) and avirulent (Xcm18) strains. Gene expression was measured by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Results are averages; error bars indicate standard deviation (n > 12). (B) Time course of oxo‐phytodienoic acid (OPDA) and JA production in response to Xcm infection. Results are averages; error bars indicate standard deviation (n > 12). Asterisks denote values significantly different from the control (C) (Student's test: *P < 0.05; **P < 0.01). All experiments (A, B) were repeated at least three times with similar results. hpi, h post‐inoculation.

Figure 3

Identification of an ethylene‐response factor (ERF)‐related transcription factor induced during Xanthomonas citri pv. malvacearum (Xcm)‐induced hypersensitive reaction (HR) in cotton plants. (A) A maximum likelihood tree representing relationships among ERF proteins from Arabidopsis thaliana (At), Oryza sativa (Os), Medicago truncatula (Mt), Solanum lycopersicum (Sly), Nicotiana tabacum (Nt), Glycine max (Gm), Vitis vinifera (Vv), Populus trichocarpa (Pt), Broussonetia papyrifera (Bp), Physcomitrella patens (Pp) and Gossypium hirsutum (Gh) plants is rooted to the APETALA2 (AP2) domain R1 (At4g36920). Bootstrap values from 100 replicates were used to assess branch support. Bootstrap values over 70 are shown. The classification by Nakano et al. (2006) is indicated by coloured accession numbers. (B) Alignment of the Gossypium hirsutum GhERF‐IIb3 with the Arabidopsis ERF transcription factor AtORA47. Underlined sequences represent the AP2 domain and the two ERF group IIb‐specific conserved motifs CMII‐1 and CMII‐3. Strictly conserved residues are highlighted in black. Numbers on the right of the alignment indicate the amino acid position for ERF transcription factors. (C) Expression levels of GhERF‐IIb3 in response to Xanthomonas virulent (Xcm20) and avirulent (Xcm18) strains, measured by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). The results are averages; error bars indicate standard deviation (n > 12). These experiments were repeated at least three times with similar results.

Figure 4

Transient overexpression of Xanthomonas citri pv. malvacearum (Xcm)‐inducible nuclear transcription factor (TF) GhERF‐IIb3 transactivates the AtAOC2 promoter in Arabidopsis thaliana cells. (A) Subcellular localization of the TF green fluorescent protein (GFP) fusions GhERF‐IIb3‐GFP in A. thaliana leaf protoplasts. GhERF‐IIb3‐GFP and NtKISa‐DsRED constructs were transformed simultaneously in protoplasts. The differential interference contrast (DIC) images are shown on the left, epifluorescence microscopic images are shown in the middle and merged images are shown on the right. (B) Schematic representation of the constructs used for promoter transactivation assays in A. thaliana protoplasts. The reporter construct consisted of the uidA gene driven by the AtAOC2 promoter [or AtAOC2 mutated promoter (pmAtAOC2) as described by Zarei et al. (2011)]. The effector constructs consisted of an expression vector carrying the CaMV35S promoter upstream of the GhERF‐IIb3, AtORA47 or GhERF‐IXa5 cDNAs. Empty effector vector was used as a negative control. The firefly luciferase (LUC) gene fused to the CaMV35S promoter served as a reference gene to correct for differences in transformation and protein extraction efficiencies. The firefly luciferase coding sequence (ffLUC) driven by the promoter CaMV35S was used as an internal control. (C) AtAOC2 promoter transactivation by the transcription factors GhERF‐IIb3 and AtORA47 in plant protoplasts. Bars represent average GUS/LUC ratios from triplicate experiments (±standard deviation) expressed relative to the vector control set at 100% (n = 6). Letters above the bars indicate distinct statistical groups as calculated by one‐way analysis of variance (ANOVA) (P < 0.01).

Figure 5

Overexpression of GhERF‐IIb3 promotes oxo‐phytodienoic acid (OPDA) and jasmonic acid (JA) accumulation in cotton plants. (A) Activation of JA‐responsive marker gene expression in cotton cotyledons overexpressing GhERF‐IIb3. (B) OPDA and JA contents in cotton cotyledons overexpressing GhERF‐IIb3. Ten‐day‐old Gossypium hirsutum plants were transformed with Agrobacterium tumefaciens carrying GhERF‐IIb3 or GFP6. At 2 days post‐transformation, gene expression and oxylipin content were determined by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) and liquid chromatography‐mass spectrometry (LC‐MS), respectively. Averages ± standard deviation (n = 24) were calculated from biological triplicates. GFP, green fluorescent protein.

Figure 6

Overexpression of GhERGF‐IIb3 induces bacterial blight resistance in cotton plants. (A) Cotton cotyledons overexpressing GhERF‐IIb3 or GFP6 as a control were inoculated at 10⁸ colony‐forming units (cfu)/mL with the virulent Xanthomonas citri pv. malvacearum (Xcm) strain (race 20). Symptom photographs at 12 days post‐inoculation (dpi). Note the hypersensitive reaction (HR)‐like symptoms in GhERFIIb3‐expressing cotyledons compared with typical yellow disease symptoms in GFP6 controls. (B) Cell death intensity at 12 dpi using water loss as proxy. (C) Kinetic evolution of cotyledon bacterial content. All experiments (B, C and D) were repeated at least three times with similar results. Averages ± standard deviation (n = 12) were calculated from biological triplicates. GFP, green fluorescent protein.