Final Report Summary - SYSA (Systemic signalling in plant – aphid interactions) Aphids are economically important phloem-feeding insect pests that do extensive damage to crops and gardens but little is known about the molecular mechanisms by which plants resist aphid attack. The reduction-oxidation (redox) status of cells, and associated regulatory and signalling processes, has long been considered to be important in the plant immune responses, particularly through interactions with plant hormone signalling pathways. Plant(phyto)hormones such as salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA) are important in the plant responses to insect herbivores such as aphids but little is known about how redox signals interact with phytohormones in order to resist infestation by aphids. The aim of this interdisciplinary project was therefore to examine the role of redox processes in plant-aphid interactions, with particular reference to the identification of systemic signalling pathways that interact with plant hormones and also to characterize these further through specific manipulations of the cellular redox balance in mutants that have defects in different specific components of the putative signalling pathways. The roles of redox processes and hormones in the response of the model plant Arabidopsis thaliana to infestation by the phloem-feeding aphid Myzus persicae were studied using state of the art approaches such as the transcriptomics and metabolite profiling. Aphid fecundity was then compared in mutants that are either lacking the major low molecular weight plant antioxidant (vitamin C, ascorbic acid) or in ABA to that measured in the wild type plants. The effects of altered ascorbate and ABA signalling on changes in SA and JA-dependent pathways upon aphid attack were then determined. Further work was also conducted in crop species particularly tobacco and potato. Whole genome microarray analysis revealed highly dynamic transcriptional responses to aphid infestation with significant differences between transcript profiles of infested and systemic leaves. These data showed aphid-dependent effects on suites of transcripts involved in redox, SA and ABA responses. The central roles of ascorbate, the ABA INSENSITIVE-4 transcription factor and OXIDATIVE SIGNAL-INDUCIBLE1 (OXI1), an important kinase in redox signalling, in plant resistance to aphids were demonstrated by the altered fecundity on respective mutants. However, ABA had a negative effect on plant resistance to aphids as did ABA INSENSITIVE-1 or REDOX RESPONSIVE TRANSCRIPTION FACTOR1. An analysis of aphid-dependent transcriptome responses in mutants defective in ascorbate accumulation (vtc2), ABA signalling (abi4) or both confirmed the importance of ABA/redox interactions in the elicitation of plant defences against aphids. These data demonstrated unequivocally that cellular redox components such as the abundance of ascorbate determine the ability of aphids to thrive and reproduce on Arabidopsis leaves. Moreover, this signalling pathway involves the ABI4 transcription factor but does not require a high level of ABA per se or the RRFT1 transcription factor for the appropriate orchestration of plant defences against aphids. These findings suggest that alterations in cellular redox state alone are sufficient to alter the binding or other properties of the ABI4 transcription factor (in the absence of changes in ABA abundance) and so trigger gene expression leading to an immune response to aphids and that this is likely to involve effects on JA signalling pathways. Hence, the redox regulation of ABI4-dependent signalling pathways is important in the regulation of aphid resistance.Other studies were performed to determine the role of protein phosphatase 2A (PP2A) in plant responses to aphids. Aphid fecundity was compared in the wild type Arabidopsis thaliana plants and in mutant lines defective in either the PP2A regulatory subunit B’γ (gamma; pp2a-b’γ) or B’ζ (zeta; pp2a-b’ζ1-1 and pp2a-b’ζ1-2) and in gamma zeta double mutants (pp2a-b’γ pp2a-b’ζ1-1) lacking both subunits. All the mutant genotypes had a highly significantly smaller leaf area than the wild-type. Moreover, aphid fecundity was significantly decreased on the pp2a-b’γ mutant relative to the wild-type, the pp2a-b’ζ1-1 and pp2a-b’ζ1-2 single mutants and the pp2a-b’γ-pp2a-b’ζ1-1 double mutant. These data demonstrate that the composition of ’subunits on the trimeric PP2A holoenzymes is important in regulating plant resistance to aphids.Plant responses to aphids were also measured in wild type and transgenic tobacco lines that have been modified to have either high or low activities of ascorbate oxidase, which an important enzyme of the apoplast/cell wall compartment of the cell. In wild-type plants, in which about 40% of the apoplastic ascorbate pool was in the reduced form (Pignocchi et al., 2003). In contrast, approximately 66% of the apoplastic ascorbate was present in the reduced form in transgenic tobacco plants with low ascorbate oxidase (TAO) activities, while only 3% of the ascorbate pool is in the reduced form in transgenic tobacco plants ectopically expressing a pumpkin (PAO) ascorbate oxidase. As discussed above, the abundance of ascorbate in leaves was shown to be important in plant resistance to aphids, with a low ascorbate level leading to an increase in aphid resistance. However, in contrast to the situation in whole leaves, low ascorbate in the apoplast cell/ wall compartment of the cell alone, as occurs in the PAO leaves was found to have no influence on aphid fecundity. In contrast, aphid fecundity was decreased in the transgenic tobacco plants with low ascorbate oxidase (TAO) activity. These data demonstrate that a high level of ascorbate in the apoplast, as is found in the TAO plants, is required for the defences that limit aphid fecundity. The abundance of ascorbate in the apoplast, which is governed by ascorbate oxidase activity, therefore exerts an influence on the ability of aphids to colonize tobacco leaves.Phloem feeding insects are the most prevalent vectors of plant viruses and also damage crops by depleting photoassimilates, manipulating growth and manipulating nutrient partitioning. Many areas of agricultural production in Europe are adversely affected by aphids. This project has produced new knowledge that contributes to the identification of factors and processes that regulate plant resistance to phloem-feeding. The results generated in this project provide essential information and molecular tools that can be used in marker assisted selection of new cultivars with improved biotic stress tolerance traits that will enhance the future predictability of crop yields. Related documents final1-publishable-summary.pdf
