The salivary gland proteome of root-galling grape phylloxera (Daktulosphaira vitifoliae Fitch) feeding on Vitis spp.

RESEARCH ARTICLE The salivary gland proteome of root-galling grape phylloxera (Daktulosphaira vitifoliae Fitch) feeding on Vitis spp. Markus W. Eitle ID1*, James C. Carolan2, Michaela Griesser1, Astrid Forneck1 1 University of Natural Resources and Life Sciences, Department of Crop Sciences, Institute of Viticulture and Pomology, Vienna, Austria, 2 Department of Biology, Maynooth University, Maynooth, Co. Kildare, Ireland a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Eitle MW, Carolan JC, Griesser M, Forneck A (2019) The salivary gland proteome of root-galling grape phylloxera (Daktulosphaira vitifoliae Fitch) feeding on Vitis spp.. PLoS ONE 14 (12): e0225881. https://doi.org/10.1371/journal. pone.0225881 Editor: Ben J. Mans, Onderstepoort Veterinary Institute, SOUTH AFRICA Received: June 27, 2019 Accepted: November 14, 2019 Published: December 17, 2019 Copyright: © 2019 Eitle et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. * Abstract The successful parasitisation of a plant by a phytophagous insect is dependent on the delivery of effector molecules into the host. Sedentary gall forming insects, such as grape phylloxera (Daktulosphaira vitifoliae FITCH, Phylloxeridae), secrete multiple effectors into host plant tissues that alter or modulate the cellular and molecular environment to the benefit of the insect. The identification and characterisation of effector proteins will provide insight into the host-phylloxera interaction specifically the gall-induction processes and potential mechanisms of plant resistance. Using proteomic mass spectrometry and in-silico secretory prediction, 420 putative effectors were determined from the salivary glands or the root-feeding D. vitifoliae larvae reared on Teleki 5C (V. berlandieri x V. riparia). Among them, 170 conserved effectors were shared between D. vitifoliae and fourteen phytophagous insect species. Quantitative RT-PCR analysis of five conserved effector candidates (protein disulfideisomerase, peroxidoredoxin, peroxidase and a carboxypeptidase) revealed that their gene expression decreased, when larvae were starved for 24 h, supporting their assignment as effector molecules. The D. vitifoliae effectors identified here represent a functionally diverse group, comprising both conserved and unique proteins that provide new insight into the D. vitifoliae–Vitis spp. interaction and the potential mechanisms by which D. vitifoliae establishes the feeding site, suppresses plant defences and modulates nutrient uptake. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Introduction Funding: The presented project was supported by a DOC research fellowship for M.W. Eitle provided by the Austrian Academy of Sciences (ÖAW) and with the Schlumberger award 2018 provided by the Robert Schlumberger foundation. Funding for Daktulosphaira vitifoliae clone Pcf genomic sequencing was provided by INRA (AIP Bioressources), BGI Biotech and i5k initiatives. The ability of phytophagous insects to feed from, or reproduce on plant hosts is dependent on the efficient modulation or evasion of plant defence systems. These defences may be constitutive or induced and can involve complex recognition and response systems that result in the release of defensive substances, targeted self-destruction of cells or even the attraction of predators to contend with the invading insect. Insects and particularly those that engage the plant host on cellular level, attempt to overcome these defences through a variety of strategies including the delivery of bioactive substances into the plant cellular environment [1]. Many of PLOS ONE | https://doi.org/10.1371/journal.pone.0225881 December 17, 2019 1 / 18 The salivary gland proteome of root-galling grape phylloxera feeding on Vitis spp. Parts of the transcriptomic resources were obtained within the 1KITE projects. The Maynooth University Q-Exactive Quantitative Mass Spectrometer was funded under the SFI Research Infrastructure Call 2012; Grant Number: 12/RI/ 2346 (3). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: Funding was provided by BGI Biotech and the i5k initiatives. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are on patents, products in development or marketed products to declare. these substances, commonly referred to as effectors, are delivered via the insect saliva directly on- or into the host tissue and ultimately determine whether the prospected interaction is compatible or incompatible. Effectors of phytophagous insects are generally small proteins or molecules that alter host cell structures and/or biological functions once they are exposed to a suitable plant tissue. Thereby single effectors may interact with several plant protein networks and modulate various host physiological traits simultaneously [2, 3]. Identifying the effector repertoire of phytophagous insect species is essential to understand the underlying host-parasite interaction and potentially leads to the development of innovative pest control strategies reducing reliance on agrochemical application [4, 5]. The identification of effector molecules within plant tissues is challenging due to their low concentration and short ephemerality due to metabolic processing. An alternative strategy is to characterise the effector-enriched salivary gland tissue, which previously resulted in the discovery of multiple insect effector proteins released by aphids [6–8], hessian flies [9], planthoppers [10, 11], whiteflies [12, 13] and thrips [14]. Significant progress in protein-based mass spectrometry and next generation sequencing technologies (e.g. RNAseq) facilitated the large-scale identification of putative insect effectors lists within insect salivary glands or saliva [15–21]. Comparative In-silico analyses of predicted effector sets across phloem-feeding aphid species identified both conserved effectors used as part of shared infestation strategies aimed to modulate common host defensive and cellular processes and unique effectors employed to establish and maintain compatible interactions in a host-specific manner [22–24]. Historically grape phylloxera (Daktulosphaira vitifoliae FITCH; Phylloxeridae) was introduced from North America into Europe in the late 19th century with devastating economic consequences for the wine industry at the time. The sedentary insect infests the Vitis spp. root system by the formation of root gall tissues, thereby inducing changes in the water, mineral and assimilate transport affecting the host vine physiology and vigor [25–27]. In addition D. vitifoliae infestation promot (...truncated)