Identification and Functional Characterization of Short Linear Motifs (SLiMs) in “Core” RxLR effector proteins from Phytophthora parasitica var nicotianae

Abstract

The growing world population together with impacts of climate change place ever-greater demands on the world food supply. On the other hand, food security is threatened by various biotic threats, including filamentous phytopathogens such as oomycetes. The genus Phytophthora is an oomycete with pronounced significance to global agriculture and food security. Chief amongst these, is the wide host range P. parasitica, which causes root and stem rot in citrus and other economically important crops of the Solanaceae family such as potato, tobacco, pepper, and tomato. The success of these pathogens is attributed to their ability to secrete effector proteins that manipulate the host immune response for disease development. Amid these proteins, RxLR (Arginine-any amino acid-Leucine-Arginine) effectors are trafficked to the inside of the host plant to cripple immune responses. A substantial advance has been provided recently through the identification of hundreds of candidate Phytophthora RxLR effector genes by means of genome sequencing and subsequent bioinformatic screens. The majority of predicted RxLR effector genes have been reported to be rapidly evolving and highly divergent. Consequently, relatively few conserved or “core” RxLR effector (CREs) genes have been identified in Phytopthora genomes. In addition, the mechanisms that these “core” effectors employ to manipulate host defence mechanisms remain poorly understood. To improve our understanding of the molecular mechanisms of action of CREs, structural analysis of these effectors is a prerequisite. Traditionally, the biological functionality of proteins including RxLRs has been strongly associated with their 3D structure. Nonetheless, recent studies have revealed the existence of “hybrid” proteins. These are proteins that consist of ordered domains and as well as intrinsically disordered regions (IDRs). These IDRs are known to encode short linear motifs (SLiMs). These are short stretches of protein sequences, about 3-10 aa long, that are crucial for biological processes through facilitating protein-protein interactions. Although intrinsic disorder has been shown to be a feature of some oomycete RxLR effectors, the contribution of SLiMs in plant disease development is still in its infancy.

In this thesis, we employed a bioinformatics pipeline, to identify secreted RxLR effectors in 11 strains of P. parasitica as well as those that are conserved among these strains. The total number of potential RxLR effectors secreted by P. parasitica strains ranges from 165 to 358. Of these, 71(21%) were found to be present in all 11 strains of P. parasitica, hence these were designated “core” RxLR effectors (CREs). We further show that the identified CREs have significantly shorter intergenic than non-conserved RxLR effector genes, and most importantly, most of the CRE genes reside in gene-dense/repeat-poor regions of the genome. This insinuates that CREs are potential candidates for breeding for durable resistance in plants by identifying their targets in plants.

Using PONDR VL-XT tool, we show the presence of intrinsically disordered regions (IDRs) in all the 71 P. parasitica CREs. We further show that the N-terminal regions (RxLR-dEER) of these effectors are significantly enriched in IDRs compared to the C-terminal regions, suggesting a potential role of disorder in effector translocation. Although the disorder content at the C-terminal region of most CREs was reduced, it is important to mention that most SLiMs are found in this region. The majority of the SLiMs identified were predicted to be implicated in protein-protein interactions. These results motivated us to study the potential role of SLiMs in detail. To this end, a candidate CRE, PpRxLR1 was used as a case study. PpRxLR1 was predicted to encode a putative SLiM at the C‐terminal end consisting of six amino acids (LWLKYQ). Classification of this motif using Eukaryotic Linear Motif (ELM) corresponded to ubiquitin associated (UBA) motif, suggesting an important role in substrate ubiquitination and consequently regulated protein degradation. Similarly, in silico prediction of PpRxLR1-host protein interaction networks revealed the association of this effector with the ubiquitin mediated proteolysis pathway as well as plant hormone signal transduction pathway. We further validated the in silico prediction results in planta where PpRxLR1 was shown to induce cell death in both Nicotianae benthamiana and N. tabacum. In addition, this study revealed that the activity of PpRxLR1 effector is mediated by two crucial amino acid residues, L102 and Y106 of a six amino acid long SLiM (LWLKYQ), encoded in this effector. To this end, a PpRxLR SLiM mutant (PpRxLR1mut) that was generated through alanine substitution of L102 and Y106 (A102 and A106) abolished the cell death inducing activity of this effector. Outstandingly, PpRxLR1mut failed to promote P. parasitica infection. Although, subcellular targeting of this effector was shown to be independent of the encoded SLiM, in planta screening of potential interactors for PpRxLR1 implicated this SLiM in mediating effector-protein interactions. Further validations are underway to confirm these screening results. Taken together, these findings open paths to delve into novel mechanisms employed by P. parasitica “core” RxLR effectors towards successful host infection. These mechanisms can be investigated further in the hunt for long-lasting and broad-spectrum breeding in plants.