Functional characterization of Pseudomonas syringae pv. actinidiae effectors AvrPto5 and HopF2

Abstract

Pseudomonas syringae pv. actinidiae (Psa) causes kiwifruit bacterial canker and was responsible for a worldwide outbreak. Four Psa biovars have been identified according to their geographical origin and genome sequence. Despite differences in the genomes of the four biovars, ten effectors were conserved in their accessory genome. Two of these, PsaNZV- 13 AvrPto5 and HopF2 were selected for investigation regarding their host protein targets in kiwifruit and non-host resistance in Nicotiana. A candidate host protein-based Y2H using PsaNZV-13 AvrPto5 was performed with kiwifruit proteins which were selected based on previously reported Y2H experiments with PtoDC3000 AvrPto1 and Arabidopsis proteins. None of the corresponding kiwifruit proteins interacted with PsaNZV-13 AvrPto5, however, phylogenetic and protein sequence analyses of PsaNZV-13 AvrPto5 demonstrated a genetic divergence from PtoDC3000 AvrPto1 which could be distinct enough to suggest PsaNZV- 13 AvrPto5 is targeting a different host protein. Intrinsic Disorder Residues (IDRs) and Molecular Recognition Feature (MoRF) investigation in PsaNZV-13 AvrPto5 and PtoDC3000 AvrPto1 revealed a higher level of IDRs (64%) and MoRF (5) in PtoDC3000 AvrPto1 compared to PsaNZV-13 AvrPto5 (IDRs: 28% and MoRF: 1), which also could explain a divergent putative function of PsaNZV-13 AvrPto5. To identify Psa effectors host target in kiwifruit, a high quality in vivo kiwifruit cDNA library was constructed in yeast. Screening of this library with PsaNZV-13 AvrPto5 found three putative positive interacting partners. One of these AcHIPP26 interacted both as bait and prey, suggesting a genuine interaction in Y2H. These proteins did not interact following Co-IP, however, suggesting either a possible disruption of the interaction complex during protein extraction or that the two proteins do not interact. Transient expression of PsaNZV-13 AvrPto5 in N. tabacum did not elicit a hypersensitive response (HR), unlike PtoDC3000 AvrPto1. PsaNZV-13 HopF2 protein sequence alignment with PtoDC3000 HopF2 showed putative ADP-ribosyltransferase (ADP-RT) (Arg72 and Asp174) and myristoylation (Gly2) residues. PsaNZV-13 HopF2 investigation in N. tabacum by transient expression revealed the HR and the ADP-RT residues of PsaNZV-13 HopF2 were critical for elicitation of HR in N. tabacum. The expression of PsaNZV-13 HopF2 in yeast cells delayed growth, which was directly correlated to the ADP-RT residues. The myristoylation residue was not critical for HR which demonstrates PsaNZV-13 HopF2 recognition does not have to occur at the plasma membrane of N. tabacum. Y2H screening revealed a subtilisin-like protease (AcSUB) as a putative target of PsaNZV-13 HopF2. In planta interaction of these proteins could not be detected due to low expression of both proteins. PsaNZV-13 HopF2 appeared to have no interaction with AtRIN4 which is the host target of PtoDC3000 HopF2 when analysed by Y2H. These results indicate a putative ADP-RT activity of PsaNZV-13 HopF2 similar to PtoDC3000 HopF2, but a different in planta target. An investigation of IDRs and MoRF in PsaNZV-13 HopF2 illustrated almost a comparable pattern of IDRs and MoRF with PtoDC3000 HopF2. Transient co-expression of PsaNZV-13 HopF2 and RNAi RIN4 constructs in N. tabacum showed an evidence for a putative role of NtRIN4 in PsaNZV-13 HopF2 induced HR. Overall, the putative ADP-RT activity of PsaNZV-13 HopF2 in N. tabacum suggests it may have a similar role as PtoDC3000 HopF2. In addition, there is a prospect to isolate the resistance gene for PsaNZV-13 HopF2 in N. tabacum.