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

Abstract

The infection of Pseudomonas syringae pv. actinidiae in kiwifruit is currently assessed by numerous methodologies, each with their own limitations. Most studies are based on either a laborious method of growth quantification of the pathogen or qualitative assessments by visual scoring following stem or cutting inoculation. Additionally, when assessing for resistance against specific pathogen effectors, confounding interactions between multiple genes in the pathogen can make mapping resistance phenotypes nearly impossible. Here we present robust alternative methods to quantify pathogen load based on rapid bacterial DNA quantification by PCR, the use of Pseudomonas fluorescens (Pfo), and a transient reporter eclipse assay, for assessing resistance conferred by isolated bacterial avirulence genes. These assays compare well with bacterial plate counts to assess bacterial colonization as a result of plant resistance activation. The DNA-based quantification, when coupled with the Pfo and reporter eclipse assays to independently identify bacterial avirulence genes, is rapid, highly reproducible, and scalable for high-throughput screens of multiple cultivars or genotypes. Application of these methodologies will allow rapid and high-throughput identification of resistant cultivars and the bacterial avirulence genes they recognize, facilitating resistance gene discovery for plant breeding programs.

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

“…Previous work showed that A . arguta plants were resistant to Psa3, associated with a quantifiable increase in ion leakage due to membrane disruption of the dying cells indicative of a hypersensitive response (HR) [ 35 ]. Leaves of A .…”

“…Using the previously described biolistic co-expression assays to measure HR-mediated reporter eclipse in AA07_03 leaves, hopZ5a was identified as the recognized effector in the hopZ5a / hopH1a effector block ( S6 Fig ). This assay, described previously, assesses whether biolistic co-delivery of an effector is able to suppress expression of a reporter GUS gene due to HR [ 35 , 42 ]. Therefore, only the single hopZ5a knockout strain was used for subsequent experiments.…”

“…syringae pv. syringae 61 was used as a positive control for HR in this assay [ 35 ]. Co- bombardment of candidate avirulence effectors hopAW1a , hopF1c , hopZ5a and avrRpm1a all demonstrated a decrease in GUS activity on A .…”

“…chinensis var. chinensis ‘Hort16A’ plantlets [ 35 ]. The GUS activity was measured 48 hours after DNA bombardment.…”

Genome Context Influences Evolutionary Flexibility of Nearly Identical Type III Effectors in Two Phytopathogenic Pseudomonads

“…Once inside the plant cells, effector proteins can disrupt plant immune responses in a variety of ways to promote bacterial growth and infection. The presence of effector proteins can also be monitored by plant immune responses through the action of R-genes, with recognition of effector protein functions leading to an overarching immune reaction termed effector triggered immunity (ETI) (Collmer et al, 2000;Jones and Dangl, 2006;Jayaraman et al, 2021). Triggering of the ETI response can quickly shut down nascent infections in resistant plant cultivars and has thus formed the basis of future plans to engineer durable crop resistance to infection through genetic modification and selective breeding (Dong and Ronald, 2019;Laflamme et al, 2020).…”