Host interactors of effector proteins of the lettuce downy mildew Bremia lactucae obtained by yeast two-hybrid screening

Abstract

Plant pathogenic bacteria, fungi and oomycetes secrete effector proteins to manipulate host cell processes to establish a successful infection. Over the last decade the genomes and transcriptomes of many agriculturally important plant pathogens have been sequenced and vast candidate effector repertoires were identified using bioinformatic analyses. Elucidating the contribution of individual effectors to pathogenicity is the next major hurdle. To advance our understanding of the molecular mechanisms underlying lettuce susceptibility to the downy mildew Bremia lactucae, we mapped physical interactions between B. lactucae effectors and lettuce candidate target proteins. Using a lettuce cDNA library-based yeasttwo-hybrid system, 61 protein-protein interactions were identified, involving 21 B. lactucae effectors and 46 unique lettuce proteins. The top ten interactors based on the number of independent colonies identified in the Y2H and two interactors that belong to gene families involved in plant immunity, were further characterized. We determined the subcellular localization of the fluorescently tagged lettuce proteins and their interacting effectors. Importantly, relocalization of effectors or their interactors to the nucleus was observed for four proteinpairs upon their co-expression, supporting their interaction in planta.

Computational Systems Biology of Alfalfa – Bacterial Blight Host-Pathogen Interactions: Uncovering the Complex Molecular Networks for Developing Durable Disease Resistant Crop

“…The effectors are deployed by the pathogenic bacteria to inhibit the plant immune response and disrupt the host cell mechanisms ( Pelgrom et al, 2020 ). In this regard, we extracted effector proteins, T3SS-hop proteins, and virulence proteins from EffectiveDB (see text footnote 3), Pseudomonas syringae Genome Resources 5 , and Pseudomonas genome DB 6 , respectively.…”

Interior design: how plant pathogens optimize their living conditions

“…Xanthomonas spp. (XopDXcv), Pseudomonas (HopF3Pph6, HopAB1Psy), and lettuce downy mildew Bremia lactucae (BLR20) have been reported to target AtERF4 (Arabidopsis), AtERF8 (Arabidopsis), and LsERF093 (closest AtERF5 lettuce orthologue), respectively (Büttner, 2016; Cao et al ., 2019; Pelgrom et al ., 2020). Interestingly, ERF4 and ERF8 possess an EAR motif that facilitates transcriptional repression instead of activation of downstream genes with roles in osmotic and drought stress adaptation (Xie et al ., 2019).…”

Functional analysis of effector proteins of the vascular wilt fungus Verticillium dahliae

“…Thus, understanding the activities of these proteins is paramount for comprehension of the virulence strategies utilized by plant pathogens to cause disease, and effectors can be utilized as probes for essential elements of host immune systems. Multiple approaches are used to gain insight into effector functions (Djamei et al, 2011;Boutemy et al, 2011;Rosli et al, 2013;Petre et al, 2015;Pelgrom et al, 2020). As interference with plant processes often involves manipulation of specific plant proteins, identification of in planta interactors of A pathogenicity determinant of V. dahliae targets auxin response factors…”

“…Given this diversity, functional characterization of effectors commonly requires multiple approaches, often employed in combination, in order to elucidate their specific activities. Such approaches include the identification of effector target(s) in planta by means of forward genetics screenings or protein-protein interaction studies (Raaymakers, 2018;Pelgrom et al, 2020), determination of subcellular localization in vivo (Petre et al, 2015;Robin et al, 2018), characterization of responses or physiological states induced by effector proteins per se on the host plant, for example in terms of transcriptome and metabolome rewiring or cellular and morphological changes in plant tissues (Djamei et al, 2011;Rosli et al, 2013;Worley et al, 2016;Redkar et al, 2017), and determination of effector protein three-dimensional structure and its potential functional implications (Boutemy et al, 2011;Sánchez-Vallet et al, 2013). Overall, these experimental paths have the potential to provide crucial information on effector activity in planta and how this may contribute to disease development.…”

“…Therefore, it has become well established that comprehensive identification of effector catalogues and determination of their modes of action is key Chapter 3 to gain mechanistic insight into the virulence programs employed by plant pathogens to foster host colonization (Lo Presti et al, 2015;Gibriel et al, 2016;Bindschedler et al, 2016;Toruño et al, 2016). Typically, functional characterization of effectors relies on multiple experimental approaches that together offer clues about their potential biochemical activities in planta (Djamei et al, 2011;Boutemy et al, 2011;Sánchez-Vallet et al, 2013;Rosli et al, 2013;Petre et al, 2015;Raaymakers, 2018;Robin et al, 2018;Pelgrom et al, 2020). Among these approaches, identification of plant proteins that act as the molecular targets of effectors is usually prioritized in effector biology studies, given that target determination provides direct evidence of the plant processes affected by the effector, thereby being instrumental for further mechanistic dissection of specific effector biochemical activities.…”