Kiwifruit defence responses to bacterial canker - the role of elicitors, cultivars, tissue type, timing & pathovars Kirstin Wurms, Tony Reglinski, Joel Vanneste, Annette Ah Chee, Allan Hardaker, Judith Bowen, Janet Phipps, Janet Yu, Deirdre Cornish, Joe Taylor, Dwayne Jensen and Janine Cooney
Host resistance role of qpcr studies Pathogen Host Environment QTL mapping correlates resistance phenotypes with specific loci on linkage groups (chromosomes) NGS/qPCR NGS shows total gene expression in response to experimental trt (costly), qpcr allows detailed examination of quantitative expression of specific genes of interest (GoI) great for mode of action studies, effect of elicitors, effect of plant tissue type and age, etc
Selection of GoI for qpcr A range of GoI was chosen to represent as many different defence response pathways and temporal stages of defence as possible
Selection of GoI for qpcr Recognition (14) e.g. RIN4, APC8 hub protein TFs e.g. WRKY40 (3) Phytohormone/signalling (12) e.g. PR1 (SA), LOX2 (JA), ABA1 (ABA) Phenylpropanoids/secondary metabolites (6) e.g. PAL, CHS, CAD, PR proteins (5) e.g PR1, PR3, PR5, β-1,3-glucosidase GoI selected based on: NGS (Psa/Hort16A), literature, and results from other kiwifruit P+D studies (qpcr, microarray, biochemical)
Overview of host gene expression studies 37 host defence genes tested Markers of phyotohormone pathways (SA/JA/ABA) developed to complement biochemical measurements 7 genotypes (Hort16A, Hayward, Gold3, Green14, and 3 pre-commercial genotypes) - reactions to the pathogen & elicitors are cultivar specific Four plant elicitors studied (ASM, MeJa, BABA, Emix) SA response predominates, ASM/Emix most effective Three tissue types (leaf above stem wound, stem local, stem systemic) localised response is strongest Psa vs. Pfm - resistance to Pfm associated with an elevated SA defence response
Cultivar-specific reactions to the pathogen & to elicitors
Elicitor mediated responses (ASM) Hort16A Hort16A plants sprayed with water or with ASM,14 d before stab inoculation with Psa (10 9 cfu/ml) or mock inoc with water Leaf above inoculation point was sampled 48 hr later for qpcr work Lesion length (mm) 60 50 40 30 20 10 0 Disease assessment 4 wks post inoculation Water/mock inoc Water/Psa ASM/mock inoc ASM/Psa
Elicitor mediated responses (ASM) Hort16A RIN4 HIRP LRR CC-NBS-LRR FLS2 Ring finger MAPKKK2 PAD4 PAL β-1,3-glucosidase TLP Water ASM Uninoc Mock Inoc Psa Uninoc Mock Inoc Psa Fold change relative to Water/Uninoc 1 2 5 > 5
Elicitor mediated responses (ASM) Gold3 RIN4 WRKY 40 AP2 ERF2 PR1 PR6 β-1,3-glucosidase LOX2 JIH1 PAL CHS CAD Water ASM Mock Inoc Psa Mock Inoc Psa Fold change relative to Water/Uninoc 1 2 5 > 5
Effect of Tissue Type on Gene Expression Hayward plantlets stab inoculated with Psa (10 9 cfu/ml). Three different sites (leaf, local stem, systemic stem) sampled 48 hr later. Defence-associated genes measured by qpcr
Effect of Tissue Type on Gene expression Many of the genes had greater induction in local tissues, e.g. PR1, PR6, AP2 ERF2 Only a few genes were more strongly induced systemically, e.g. WRKY40
Temporal Effect of Gene Expression Gold3 plants sprayed with water or ASM 7d before stab inoculation with Psa (10 9 cfu/ml) or water (mock inoc). Stem & leaf above inoc point sampled 0, 3, 6, 24 and 48 h later Same exp repeated in Hayward and measurements are underway
Psa vs. Pfm Pfm causes less disease, and a greater induction of SA and SA-based defences than Psa
Summary Host defence gene expression in response to Psa infection and to elicitor treatment is cultivar specific, so elicitor selection and use needs to be optimised for each cultivar. Of the elicitors studied (ASM, MeJa, BABA, Emix) ASM/Emix most effective, with markers of the SA defence response predominating, e.g. PR1, PR6, PAD4, β-1,3- glucosidase Three tissue types (leaf above stem wound, stem local, stem systemic) localised response is strongest e.g. PR1, PR6, AP2 ERF2
Summary Temporal expression experiments have shown that some genes involved in recognition and signalling are expressed within 3-6 hrs, while expression of PR proteins, such as PR1 occurs later (24 hrs). Elicitors can prime the plant enabling it to respond more quickly and/or to a greater extent upon pathogen ingress e.g. CAD, ferronia. Psa vs. Pfm - resistance to Pfm associated with an elevated SA concentration, and increased expression of SA defence genes e.g. PR1, β-1,3-glucosidase Some of the most promising markers to date include β-1,3-glucosidase, PR1, JAR1, JIH1, ABA1, RIN4. This list is always changing as we are continually developing new markers
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