Short Linear Motifs (SLiMs) in "Core" RxLR Effectors of Phytophthora parasitica var. nicotianae: a Case of PpRxLR1 Effector.

Oomycetes of the genus Phytophthora encompass several of the most successful plant pathogens described to date. The success of infection by Phytophthora species is attributed to the pathogens' ability to secrete effector proteins that alter the host's physiological processes. Structural analyses of effector proteins mainly from bacterial and viral pathogens have revealed the presence of intrinsically disordered regions that host short linear motifs (SLiMs). These motifs play important biological roles by facilitating protein-protein interactions as well as protein translocation. Nonetheless, SLiMs in Phytophthora species RxLR effectors have not been investigated previously and their roles remain unknown. Using a bioinformatics pipeline, we identified 333 candidate RxLR effectors in the strain INRA 310 of Phytophthora parasitica. Of these, 71 (21%) were also found to be present in 10 other genomes of P. parasitica, and hence, these were designated core RxLR effectors (CREs). Within the CRE sequences, the N terminus exhibited enrichment in intrinsically disordered regions compared to the C terminus, suggesting a potential role of disorder in effector translocation. Although the disorder content was reduced in the C-terminal regions, it is important to mention that most SLiMs were in this terminus. PpRxLR1 is one of the 71 CREs identified in this study, and its genes encode a 6-amino acid (aa)-long SLiM at the C terminus. We showed that PpRxLR1 interacts with several host proteins that are implicated in defense. Structural analysis of this effector using homology modeling revealed the presence of potential ligand-binding sites. Among key residues that were predicted to be crucial for ligand binding, L102 and Y106 were of interest since they form part of the 6-aa-long PpRxLR1 SLiM. In silico substitution of these two residues to alanine was predicted to have a significant effect on both the function and the structure of PpRxLR1 effector. Molecular docking simulations revealed possible interactions between PpRxLR1 effector and ubiquitin-associated proteins. The ubiquitin-like SLiM carried in this effector was shown to be a potential mediator of these interactions. Further studies are required to validate and elucidate the underlying molecular mechanism of action. IMPORTANCE The continuous gain and loss of RxLR effectors makes the control of Phytophthora spp. difficult. Therefore, in this study, we endeavored to identify RxLR effectors that are highly conserved among species, also known as "core" RxLR effectors (CREs). We reason that these highly conserved effectors target conserved proteins or processes; thus, they can be harnessed in breeding for durable resistance in plants. To further understand the mechanisms of action of CREs, structural dissection of these proteins is crucial. Intrinsically disordered regions (IDRs) that do not adopt a fixed, three-dimensional fold carry short linear motifs (SLiMs) that mediate biological functions of proteins. The presence and potential role of these SLiMs in CREs of Phytophthora spp. have been overlooked. To our knowledge, we have effectively identified CREs as well as SLiMs with the potential of promoting effector virulence. Together, this work has advanced our comprehension of Phytophthora RxLR effector function and may facilitate the development of innovative and effective control strategies.

Isolation of endophytic bacteria from the leaves of Anredera cordifolia CIX1 for metabolites and their biological activities.

BACKGROUND: Endophytes, especially those that are found from ethnopharmacologically noteworthy medicinal plants have attracted attention due to their diverse bioactive metabolites of pharmacological importance. METHODS: This study aimed at isolating endophytic bacterium from the leaves of Anredera cordifolia CIX1 for its bioactive metabolites. The endophytic isolates were identified by 16S rRNA sequence and investigated for antibiotic sensitivity using different antibiotics. The secondary metabolites were evaluated for antibacterial activity against four bacterial strains. The 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2'-azinobis (3- ethylbenzothiazoline-6-sulfonic acid) (ABTS) methods were used to assess their scavenging activities. The chemical components were analysed by gas chromatography-mass spectrometry (GC-MS). RESULTS: Out of 13 isolates, Isolate 1 was identified as Pseudomonas aeruginosa CP043328.1. It was resistant to clindamycin, ertapenem, penicillin G, amoxicillin, cephalothin and kanamycin but sensitive to imipenem, meropenem, and gentamycin. Its extract demonstrated antibacterial activity with minimum inhibitory concentration value of 0.098 against Bacillus cereus (ATCC 10102) and Staphylococcus aureus (ATCC 25925) and 0.391 mg/ml against Escherichia coli (ATCC 25922) and Proteus mirabilis (ATCC 25933). The extract revealed DPPH and ABTS scavenging activities with half maximal inhibitory concentration value of 0.650 mg/ml and 0.15 mg/ml, respectively. The GC-MS revealed a total of 15 compounds with diisooctyl phthalate (50.51%) and [1, 2, 4] oxadiazole, 5-benzyl-3 (10.44%) as major components. CONCLUSIONS: P. aeruginosa CP043328.1 produced secondary metabolites with antibacterial and antioxidant activities.