A Putative Apoplastic Effector of Clavibacter capsici, ChpG_Cc as Hypersensitive Response and Virulence (Hrv) Protein in Plants

Abstract

Clavibacter bacteria use secreted apoplastic effectors, such as putative serine proteases, for virulence in host plants and for hypersensitive response (HR) induction in nonhost plants. Previously, we have shown that Clavibacter capsici ChpGCc is important for the necrosis development in pepper leaves. Here we determine the function of ChpGCc, along with three paralogous proteins, for HR induction in the apoplastic space of a nonhost plant, Nicotiana tabacum. The full-length and signal peptide–deleted (ΔSP) mature forms of all proteins fused with the tobacco PR1b signal sequence were generated. The full-length and ΔSP forms of ChpGCc and only the ΔSP forms of ChpECc and Pat-1Cc, but none of the ChpCCc triggered HR. Based on the predicted protein structures, ChpGCc carries amino acids for a catalytic triad and a disulfide bridge in positions like Pat-1Cm. Substituting these amino acids of ChpGCc with alanine abolished or reduced HR-inducing activity. To determine whether these residues are important for necrosis development in pepper, alanine-substituted chpGCc genes were transformed into the C. capsici PF008ΔpCM1 strain, which lacks the intact chpGCc gene. The strain with any variants failed to restore the necrosis-causing ability. These results suggest that ChpGCc has a dual function as a virulence factor in host plants and an HR elicitor in nonhost plants. Based on our findings and previous results, we propose Clavibacter apoplastic effectors, such as ChpGCc, Pat-1Cm, Chp-7Cs, and ChpGCm, as hypersensitive response and virulence (Hrv) proteins, displaying phenotypic similarities to the hypersensitive response and pathogenicity (Hrp) proteins found in Gram-negative bacteria.

Allelic variations in the chpG effector gene within Clavibacter michiganensis populations determine pathogen host range

“…The S231A substitution within the serine hydrolase catalytic triad in ChpG homologs of Cm and C . capsici resulted in a loss of activity regarding HR elicitation and virulence [ 39 , 46 ]. This suggests that ChpG is an active serine hydrolase and is unlikely to function as a pseudoprotease [ 76 ].…”

“…However, their function and plant targets are unknown. In addition, serine protease activity has yet to be demonstrated in proteins of the Chp/Pat-1 family; this activity has only been inferred based on protein similarity and the observation that disruption of the serine hydrolase catalytic triad abolishes their ability to contribute to virulence or induce HR-like cell death in non-host plants [ 36 – 39 ].…”

“…A chpG inactivation mutant in the background of the Cm model strain Cm 101 cannot cause HR and is fully pathogenic in numerous eggplant varieties [ 46 ]. Introduction of chpG into the Cm 101 chpG inactivation mutant restored immune recognition in eggplant, while introduction of a chpG variant containing a point mutation that results in the substitution of the serine residue within the predicted Ser/His/Asp catalytic triad at position 231 [ 33 , 39 , 46 ] to alanine failed to do the same [ 46 ]. This indicates that ChpG is a recognized effector, potentially through a byproduct of its putative catalytic activity, and therefore, its recognition in eggplant is likely to follow the gene-for-gene model.…”