Proteomic Analysis and Functional Validation of a Brassica oleracea Endochitinase Involved in Resistance to Xanthomonas campestris

Abstract

Black rot is a severe disease caused by the bacterium Xanthomonas campestris pv. campestris (Xcc), which can lead to substantial losses in cruciferous vegetable production worldwide. Although the use of resistant cultivars is the main strategy to control this disease, there are limited sources of resistance. In this study, we used the LC-MS/MS technique to analyze young cabbage leaves and chloroplast-enriched samples at 24 h after infection by Xcc, using both susceptible (Veloce) and resistant (Astrus) cultivars. A comparison between susceptible Xcc-inoculated plants and the control condition, as well as between resistant Xcc-inoculated plants with the control was performed and more than 300 differentially abundant proteins were identified in each comparison. The chloroplast enriched samples contributed with the identification of 600 additional protein species in the resistant interaction and 900 in the susceptible one, which were not detected in total leaf sample. We further determined the expression levels for 30 genes encoding the identified differential proteins by qRT-PCR. CHI-B4 like gene, encoding an endochitinase showing a high increased abundance in resistant Xcc-inoculated leaves, was selected for functional validation by overexpression in Arabidopsis thaliana. Compared to the wild type (Col-0), transgenic plants were highly resistant to Xcc indicating that CHI-B4 like gene could be an interesting candidate to be used in genetic breeding programs aiming at black rot resistance.

Advances in Multi-Omics Approaches for Molecular Breeding of Black Rot Resistance in Brassica oleracea L.

“…Also, decreased abundance of ubiquitin (to stop the bacteria from using ubiquitination pathway) and malate dehydrogenase (to reduce energy metabolism in the early stage of infection) were found, playing important roles in the resistance mechanisms against Xcc. Proteome analysis of Xccinfected young cabbage leaves and chloroplast-enriched samples of both the susceptible and resistant cultivars revealed the differential abundance of photosynthesis-related proteins in both resistance and susceptible interactions (Santos C. et al, 2019). As…”

“…It has been shown that increased abundance of proteins involved with ABA may favour susceptibility ( Kim et al, 2011 ; Desclos-Theveniau et al, 2012 ). The ABA signalling pathway is manipulated by a type III effector (AvrXccC8004) of Xcc , thereby increasing the ABA levels ( Ho et al, 2013 ) and proteins responsive to ABA ( Santos C. et al, 2019 ) during the infection by Xcc . ABA along with ROS and elicitors of plant defence may stimulate Ca 2+ influx ( Tortosa et al, 2019 ) which may increase Ca 2+ in guard cells prompting stomatal closure ( Klüsener et al, 2002 ).…”

“…Xcc infection promotes changes in the secondary and primary metabolism in the host to induce defence programs affecting growth and development ( Vega-Álvarez et al, 2021 ). The changes mostly include down-regulation of proteins involved in photosynthesis ( Ribeiro et al, 2018 ; Santos C. et al, 2019 ). An earlier study ( Villeth et al, 2016 ) had shown a clear reduction in the abundance of proteins involved in energetic metabolism in susceptible interaction with an opposite behaviour in the resistance interaction.…”

“…Also, decreased abundance of ubiquitin (to stop the bacteria from using ubiquitination pathway) and malate dehydrogenase (to reduce energy metabolism in the early stage of infection) were found, playing important roles in the resistance mechanisms against Xcc . Proteome analysis of Xcc -infected young cabbage leaves and chloroplast-enriched samples of both the susceptible and resistant cultivars revealed the differential abundance of photosynthesis-related proteins in both resistance and susceptible interactions ( Santos C. et al, 2019 ). As expected, most of the photosynthesis-related proteins showed decreased abundance (18%) in the resistance interactions, whereas in the susceptible interaction, increased abundance of proteins was observed consistent with the previous result obtained by Ribeiro et al (2018) .…”

Network Topological Analysis for the Identification of Novel Hubs in Plant Nutrition

“…Instead, fewer studies rely on protein–protein interaction (PPI) networks, mainly due to the lack of accurate plant models ( Di Silvestre et al, 2018 ). Nevertheless, the number of studies focusing on high-throughput profiling of plant proteomes ( Vigani et al, 2017 ; Castano-Duque et al, 2018 ; Santos et al, 2019 ), on experimental identification of PPIs ( Senkler et al, 2017 ; Osman et al, 2018 ; Huo et al, 2020 ) or their computational prediction ( Ding and Kihara, 2019 ) are recently increasing. The computational prediction of PPI is usually inferred by transferring interactions from model plant orthologs, like Arabidopsis thaliana.…”