Molecular characterization of XopAG effector AvrGf2 from Xanthomonas fuscans ssp. aurantifolii in grapefruit

Abstract

Xanthomonas fuscans ssp. aurantifolii group C strains exhibit host specificity on different citrus species. The strains possess a type III effector, AvrGf2, belonging to the XopAG effector gene family, which restricts host range on citrus. We dissected the modular nature and mode of action of AvrGf2 in grapefruit resistance. XopAG effectors possess characteristic features, such as a chloroplast localization signal, a cyclophilin-binding domain characteristic amino acid sequence motif (GPLL) and a C-terminal domain-containing CLNAxYD. Mutation of GPLL to AASL in AvrGf2 abolished the elicitation of the hypersensitive response (HR), whereas mutation of only the first amino acid to SPLL delayed the HR in grapefruit. Yeast two-hybrid experiments showed strong interaction of AvrGf2 with grapefruit cyclophilin (GfCyp), whereas AvrGf2-SPLL and AvrGf2-AASL mutants showed weak and no interaction, respectively. Molecular modelling and in silico docking studies for the cyclophilin-AvrGf2 interaction predicted the binding of citrus cyclophilins (CsCyp, GfCyp) to hexameric peptides spanning the cyclophilin-binding domain of AvrGf2 and AvrGf2 mutants (VAGPLL, VASPLL and VAAASL) with affinities equivalent to or better than a positive control peptide (YSPSA) previously demonstrated to bind CsCyp. In addition, the C-terminal domain of XopAG family effectors contains a highly conserved motif, CLNAxYD, which was identified to be crucial for the induction of HR based on site-directed mutagenesis (CLNAxYD to CASAxYD). Our results suggest a model in which grapefruit cyclophilin promotes a conformational change in AvrGf2, thereby triggering the resistance response.

Recent advances in the understanding of Xanthomonas citri ssp. citri pathogenesis and citrus canker disease management

“…Mutagenesis of the GPLL site in avrGf2 resulted in a loss of the ability to elicit an HR (Gochez et al, 2016). Molecular modelling and in silico docking studies for the Cyp-avrGf2 interaction predicted the binding of citrus Cyp, which accelerates protein folding through a peptidylprolyl isomerase activity that catalyses a cis-trans isomerization of proline (Pro) peptide bonds (Gochez et al, 2016). The proposed model for the mode of action of avrGf2 in citrus suggests an important role for Cyp as a catalyst.…”

“…The XopAG homologue, avrGf2, which also elicits an HR in grapefruit, has been isolated from Xcc-C (Gochez et al, 2015). A Cyp-binding site (GPLL) present in avrGf2 (and in every member of the XopAG effector family) was essential for the expression of an HR (Gochez et al, 2016). Mutagenesis of the GPLL site in avrGf2 resulted in a loss of the ability to elicit an HR (Gochez et al, 2016).…”

“…A Cyp-binding site (GPLL) present in avrGf2 (and in every member of the XopAG effector family) was essential for the expression of an HR (Gochez et al, 2016). Mutagenesis of the GPLL site in avrGf2 resulted in a loss of the ability to elicit an HR (Gochez et al, 2016). Molecular modelling and in silico docking studies for the Cyp-avrGf2 interaction predicted the binding of citrus Cyp, which accelerates protein folding through a peptidylprolyl isomerase activity that catalyses a cis-trans isomerization of proline (Pro) peptide bonds (Gochez et al, 2016).…”

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

“…The type III effector AvrGf2, secreted by Xfa , is responsible for triggering HDR in Citrus spp., except in C. aurantifolia . AvrGf2 belongs to the XopAG effector family which is present in several xanthomonads, including Xcc (Gochez et al ., ). To further determine whether the NHR triggered by Xcc involves type III effector proteins, the hrcV ‐deficient mutant strain of Xcc 8004 ( Xcc Δ hrcV ), defective in the T3S system (Cerutti et al ., ), was assayed.…”

“…This response is associated with an HR at the site of bacterial infection that limits pathogen growth (Brunings and Gabriel, ; Chiesa et al ., ; Gochez et al ., ). Recently, an Xfa effector (AvrGf2/XopAG) has been identified as responsible for triggering the HR (Gochez et al ., , ), suggesting that this HDR is predominantly regulated by an ETI. However, as yet, no resistance ( R ) genes have been identified in citrus.…”