Effector loss drives adaptation of Pseudomonas syringae pv. actinidiae biovar 3 to Actinidia arguta

Abstract

A pandemic isolate of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has devastated kiwifruit orchards growing cultivars of Actinidia chinensis. In contrast, A. arguta (kiwiberry) is not a host of Psa3. Resistance is mediated via effector-triggered immunity, as demonstrated by induction of the hypersensitive response in infected A. arguta leaves, observed by microscopy and quantified by ion-leakage assays. Isolates of Psa3 that cause disease in A. arguta have been isolated and analyzed, revealing a 51 kb deletion in the exchangeable effector locus (EEL). This natural EEL-mutant isolate and strains with synthetic knockouts of the EEL were more virulent in A. arguta plantlets than wild-type Psa3. Screening of a complete library of Psa3 effector knockout strains identified increased growth in planta for knockouts of four effectors–AvrRpm1a, HopF1c, HopZ5a, and the EEL effector HopAW1a –suggesting a resistance response in A. arguta. Hypersensitive response (HR) assays indicate that three of these effectors trigger a host species-specific HR. A Psa3 strain with all four effectors knocked out escaped host recognition, but a cumulative increase in bacterial pathogenicity and virulence was not observed. These avirulence effectors can be used in turn to identify the first cognate resistance genes in Actinidia for breeding durable resistance into future kiwifruit cultivars.

Contrasting effector profiles between bacterial colonisers of kiwifruit reveal redundant roles and interplay converging on PTI-suppression and RIN4

“…Asterisks indicate statistically significant differences from Welch's t-test between the indicated strain and wild-type Psa3 V-13, where p≤.001 (***), or p≤.0001 (****); not significant (ns). hopH1a,avrPto1b,avrRpm1a,and tEEL (hopF1a,hopAF1b,hopD2a and hopF1e) This study Psa3 V-13 ΔhrcC deleted hrcC (Colombi et al, 2017) Psa3 V-13 ΔCEL deleted hopN1a, shcM, hopM1f, hrpW1, shcE and avrE1d (Jayaraman et al, 2020) Psa3 V-13 ΔsEEL deleted hopAW1a, hopF1e (and shcF), hopD2a, hopAF1b, and hopF1a (and shcF) (Hemara et al, 2022) Psa3 V-13 ΔfEEL deleted avrD, hopF4a (and shcF), avrB2b, hopAW1a, hopF1e (and shcF), hopD2a, hopAF1b, and hopF1e (and shcF) (Hemara et al, 2022) Psa3 V-13 ΔxEEL deleted hopQ1a, hopD1a, avrD, hopF4a…”

“…CC-BY-NC 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in evolved to recognise at least three of these effectors, and their deletion leads to an increase in fitness (Hemara et al, 2022). This illustrates the active role being played by these RIN4-interacting effectors in the evolution of Psa and kiwifruit germplasm near the likely point of origin of Psa as a species (McCann et al, 2017).…”

“…To make the Pfm LV-5 Δ hopA1a , Δ hopE1a , or Δ hopA1a /Δ hopE1a mutants, or Psa3 V-13 Δ hopH1a , Δ hopQ1a /Δ hopD1a , Δ hopS2b /Δ hopAZ1a , Δ CEL /Δ xEEL , Δ CEL /Δ hopS2b /Δ hopAZ1a , Δ CEL /Δ xEEL /Δ hopS2b /Δ hopAZ1a , Δ hopH1a /Δ hopZ5a /Δ avrPto1b , Δ hopH1a /Δ hopZ5a /Δ avrPto1b /Δ avrRpm1a and Δ hopH1a /Δ hopZ5a /Δ avrPto1b /Δ avrRpm1a /Δ tEEL mutants, methodologies similar to that used for Psa3 V-13 multi-effector knockouts described earlier were used (Hemara et al ., 2022). Briefly, for each multi-effector knockout, a selected Psa3 V-13 or Pfm LV-5 strain was transformed by electroporation with the relevant pΔ(T3E) construct and transconjugants were selected on LB plates with nitrofurantoin, cephalexin, and kanamycin.…”

“…(and shcF), hopD2a, hopAF1b, and hopF1a (and shcF) (Hemara et al, 2022) Psa3 V-13 ΔtEEL deleted hopF1e (and shcF), hopD2a, hopAF1b, and hopF1a (and shcF) (Hemara et al, 2022) Psa3 V-13 ΔhopZ5a/ΔhopH1a Double mutant; deleted hopZ5a and hopH1a (Hemara et al, 2022) Psa3 V-13 ΔhopAM1-1/ΔhopAM1-2 Double mutant; deleted hopAM1a-1 and hopAM1a-2 (Hemara et al, 2022) Psa3 V-13 ΔhopQ1 deleted hopQ1a (Hemara et al, 2022) Psa3 V-13 ΔhopD1 deleted hopD1a (Hemara et al, 2022) Psa3 V-13 ΔhopI1 deleted hopI1c (Hemara et al, 2022) Psa3 V-13 ΔhopY1 deleted hopY1b (Hemara et al, 2022) Psa3 V-13 ΔavrRpm1a deleted avrRpm1a (Hemara et al, 2022) Psa3 V-13 ΔhopW1c deleted hopW1c (Hemara et al, 2022) Psa3 V-13 ΔhopBN1a deleted hopBN1a (and shcF) (Hemara et al, 2022) . CC-BY-NC 4.0 International license perpetuity.…”

“…CC-BY-NC 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in Psa3 V-13 ΔhopAZ1a deleted hopAZ1a (Hemara et al, 2022) Psa3 V-13 ΔhopF1c deleted hopF1c (and shcF) (Hemara et al, 2022) Psa3 V-13 ΔhopAU1a deleted hopAU1a (Hemara et al, 2022) Psa3 V-13 ΔhopBP1a deleted hopBP1a (Hemara et al, 2022) Psa3 V-13 ΔhopAS1b deleted hopAS1b (Hemara et al, 2022) Psa3 V-13 ΔavrPto1b deleted avrPto1b (Hemara et al, 2022) Psa3 V-13 ΔhopS2b deleted hopS2b (and shcS2) (Hemara et al, 2022) Psa3 V-13 ΔhopZ5a deleted hopZ5a (Hemara et al, 2022) Psa3 V-13 + pBBR1MCS-5 Plasmid-complemented with empty vector (EV) (Hemara et al, 2022) Psa3 V-13 ΔhrcC + pBBR1MCS-5 Plasmid-complemented with empty…”