AvrE1 and HopR1 from Pseudomonas syringae pv. actinidiae are additively required for full virulence on kiwifruit

Abstract

21Pseudomonas syringae pv. actinidiae ICMP 18884 biovar 3 (Psa3) produces 22 necrotic lesions during infection of its kiwifruit host. Bacterial growth in planta 23 and lesion formation are dependent upon a functional type III secretion system 24 (T3S), which translocates multiple effector proteins into host cells. Associated 25 with the T3S locus is the conserved effector locus (CEL), which has been 26 characterised and shown to be essential for the full virulence in other P. syringae 27 pathovars. Two effectors at the CEL, hopM1 and avrE1, as well as an avrE1-related 28 non-CEL effector, hopR1, have been shown to be redundant in the model pathogen 29 P. syringae pv. tomato DC3000 (Pto), a close relative of Psa. However, it is not 30 known whether CEL-related effectors are required for Psa pathogenicity. The Psa3 31 allele of hopM1, and its associated chaperone, shcM, have diverged significantly 32 from their orthologs in Pto. Furthermore, the CEL effector hopAA1-1, as well as a 33 related non-CEL effector, hopAA1-2, have both been pseudogenised. We have 34shown that HopM1 does not contribute to Psa3 virulence due to a truncation in 35 shcM, a truncation conserved in the Psa lineage, likely due to the need to evade 36 HopM1-triggered immunity in kiwifruit. We characterised the virulence 37 contribution of CEL and related effectors in Psa3 and found that only avrE1 and 38 hopR1, additively, are required for in planta growth and lesion production. This is 39 unlike the redundancy described for these effectors in Pto and indicates that these 40

Rapid Methodologies for AssessingPseudomonas syringaepv.actinidiaeColonization and Effector-Mediated Hypersensitive Response in Kiwifruit

“…The inoculum was poured off the plantlets, and the pottles were resealed with lids and were kept under plant growth conditions until sampled. In planta bacterial growth was assessed as described previously described (Jayaraman et al, 2020). Briefly, four leaf discs per pseudobiological replicate (with four replicates per treatment, per timepoint), randomly sampled using a 1-cm diameter cork-borer from three plants in a single pottle, were harvested at 2 h (day 0), day 6, and day 12 post-inoculation.…”

“…chinensis 'Hort16A' triggers defence gene expression when infected with Psa3. Defense gene expression in 'Hort16A' or AA07_03 in response to Psa3 infection Leaves from plantlets of 'Hort16' and AA07_03 were vacuum-infiltrated with mM MgSO 4 buffer (mock), Psa3 wildtype (WT), or type III secretion system mutant (ΔhrcC) at 833~10 8 colony forming units; CFU/mL and harvested at 20 hours post-infiltration for defense gene expression (AcWRKY70a; Acc03057.1, or its homolog in A. arguta) determined from extracted RNA by quantitative polymerase chain reaction as done previously (Jayaraman et al, 2020). Expression for Leaf discs from 'Hort16A' plantlets were vacuuminfiltrated with Pfo Pf0-1 wildtype (WT) or type III secretion system carrying mutant (T3S) carrying empty vector (EV) or putative avirulence effector genes avrRpm1PmaM2, avrRpt2PtoT1, avrPphBPph1448A, or hopA1Psy61 inoculum at ~108 colony forming units; CFU/mL, and electrical conductivity due to HRassociated electrolyte leakage measured at selected timepoints over 48 h. Error bars represent the standard errors of the means calculated from five biological replicates.…”

“…Quantitative PCR (qPCR) was carried out on an Illumina Eco™ Real-Time PCR machine using the EvaGreen SsoFast™ qPCR mix (BioRad, NZ). Defence gene expression (AcWRKY70a or AaWRKY70a; (Jayaraman et al, 2020)) was determined by normalization to internal AcEF1α ('Hort16A') or AaEF1α (AA07_03) expression, relative to levels in mock. Primers used for qPCR are listed in Table S1.…”

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

“…Briefly, flanking regions 1kb upstream (UP) and 1kb downstream (DN) of the effectors of interest were PCRamplified with UP-R and DN-F cloning primers carrying an inserted XbaI site (Table S1), digested with XbaI restriction enzyme (New England Biolabs/NEB, MA, USA), and ligated to form a 2 kb knockout fragment. This 2 kb fragment was subsequently cloned into the Eco53kI restriction enzyme (NEB) site of pK18B-E (Jayaraman et al, 2020). The knockout fragment sequence and quality were verified by sequencing using M13F and M13R primers (Macrogen, South Korea).…”

Characterization of Virulence Function of Pseudomonas cichorii Avirulence Protein E1 (AvrE1) during Host Plant Infection

“…In the bacterial canker disease causing P. syringae pv. actinidiae, the cooperation of AvrE1 and HopR1 was required to foster in planta bacterial growth and lesion production in kiwi fruits (Jayaraman et al, 2020).…”