Resistance to citrus canker induced by a variant of Xanthomonas citri ssp. citri is associated with a hypersensitive cell death response involving autophagy‐associated vacuolar processes

Abstract

Xanthomonas citri ssp. citri (X. citri) is the causal agent of Asiatic citrus canker, a disease that seriously affects most commercially important Citrus species worldwide. We have identified previously a natural variant, X. citri A , that triggers a host-specific defence response in Citrus limon. However, the mechanisms involved in this canker disease resistance are unknown. In this work, the defence response induced by X. citri A was assessed by transcriptomic, physiological and ultrastructural analyses, and the effects on bacterial biofilm formation were monitored in parallel. We show that X. citri A triggers a hypersensitive response associated with the interference of biofilm development and arrest of bacterial growth in C. limon. This plant response involves an extensive transcriptional reprogramming, setting in motion cell wall reinforcement, the oxidative burst and the accumulation of salicylic acid (SA) and phenolic compounds. Ultrastructural analyses revealed subcellular changes involving the activation of autophagy-associated vacuolar processes. Our findings show the activation of SA-dependent defence in response to X. citri A and suggest a coordinated regulation between the SA and flavonoid pathways, which is associated with autophagy mechanisms that control pathogen invasion in C. limon. Furthermore, this defence response protects C. limon plants from disease on subsequent challenges by pathogenic X. citri. This knowledge will allow the rational exploitation of the plant immune system as a biotechnological approach for the management of the disease.

Plant responses underlying nonhost resistance of Citrus limon against Xanthomonas campestris pv. campestris

“…Green fluorescent protein ( GFP )‐labelled strains of Xcc and Xfa were used to analyse the phenotype developed over a 16‐day post‐inoculation (dpi) period. A pathogenic strain of X. citri was used as reference for canker development (Roeschlin et al ., ). The results obtained for ‘Eureka’ are shown in Fig.…”

PthA4AT, a 7.5‐repeats transcription activator‐like (TAL) effector from Xanthomonas citri ssp. citri, triggers citrus canker resistance

“…Escherichia coli strains DH5α and BL21 and Agrobacterium tumefaciens GV3101 were grown in Luria‐Bertani (LB) medium at 37 °C and 28 °C, respectively. Recombinants plasmids were introduced into the X. citri and Agrobacterium strains by electroporation (Roeschlin et al ., ). When required, the antibiotics ampicillin (100 µg/mL), kanamycin (50 µg/mL), rifampicin (50 µg/mL) or spectinomycin (100 µg/mL) were added to the growth media.…”

“…For pathogenicity assays on Citrus spp., new shoots were selected according to Roeschlin et al . (). Bacterial suspensions of 10 7 or 10 9 colony‐forming units (cfu) per millilitre were prepared in 10 mM MgCl 2 and inoculated by infiltration or cotton swab onto 20‐day‐old leaves of the new shoots, respectively.…”

“…Symptoms progression was phenotypically monitored using an MVX10 stereomicroscope as reported in Roeschlin et al . () and conductivity was measured according to Chiesa et al . ().…”

“…interactions in which the effector acts as an avirulence factor (Chen et al ., ; Deng et al ., ; Khalaf et al ., ; Lee et al ., ). Recently, we have isolated a X. citri strain ( X. citri A T ) that triggers a host‐specific defence response in Citrus limon that is associated with the interference of biofilm development and arrest of bacterial growth (Chiesa et al ., ; Roeschlin et al ., ). The occurrence of an oxidative burst, the accumulation of salicylic acid and phenolic compounds, and the hypersensitive response (HR) together suggest that it is an ETI response.…”