Transcriptome analysis of Phytophthora cactorum infecting strawberry identified RXLR effectors that induce cell death when transiently expressed in Nicotiana benthamiana

Abstract

Phytophthora cactorum is a plant pathogenic oomycete that causes crown rot in strawberry leading to significant economic losses every year. To invade the host, P. cactorum secretes an arsenal of effectors that can manipulate host physiology and impair its defense system promoting infection. A transcriptome analysis was conducted on a susceptible wild strawberry genotype (Fragaria vesca) 48 hours post inoculation with P. cactorum to identify effectors expressed during the early infection stage. The analysis revealed 4,668 P. cactorum genes expressed during infection of F. vesca. A total of 539 secreted proteins encoded by transcripts were identified, including 120 carbohydrate-active enzymes, 40 RXLRs, 23 proteolytic enzymes, nine elicitins, seven cysteine rich proteins, seven necrosis inducing proteins and 216 hypothetical proteins with unknown function. Twenty of the 40 RXLR effector candidates were transiently expressed in Nicotiana benthamiana using agroinfiltration and five previously unreported RXLR effector genes (Pc741, Pc8318, Pc10890, Pc20813, and Pc22290) triggered cell death when transiently expressed. The identified cell death inducing RXLR effectors showed 31-66% identity to known RXLR effectors in different Phytophthora species having roles in pathogenicity including both activation and suppression of defense response in the host. Furthermore, homology analysis revealed that these cell death inducing RXLR effectors were highly conserved (82 - 100% identity) across 23 different strains of P. cactorum originating from apple or strawberry.

Keywords: RNA-Seq; agroinfiltration; disease resistance response; host-pathogen interaction; oomycete.

Figure 1

Gene ontology (GO) classification and Kyoto encyclopedia of genes and genome (KEGG) analysis of proteins encoded by transcripts of Phytophthora cactorum (accession GCA_003287315.1) detected in the rhizome (crown) of the susceptible Fragaria vesca NCGR1218, 48 hours after inoculation. The GO terms and KEGG pathways were categorized using the STRING V11.5 database (Szklarczyk et al., 2021). The top 10 most represented terms in each of the GO categories cellular component, molecular function, and biological process, as well as KEGG pathways are shown.

Figure 2

Secreted Phytophthora cactorum proteins with similarity to proteins that influence pathogen host interaction (PHI), based on the PHI-base (Urban et al., 2022). The proteins are deduced from transcripts detected in the rhizome (crown) of a susceptible Fragaria vesca genotype (NCGR1218), 48 hours after inoculation with P. cactorum. The PHI-phenotypes are resulting from a mutation or altered expression of the specific gene in the pathogen while ‘plant avirulence determinant’ represents an effector required for recognition of a pathogen by the resistant host. Pc is the abbreviated form of PC110_g in the gene identifier and is obtained from P. cactorum accession GCA_003287315.1. The gene IDs in bold are assigned to more than one PHI-phenotype.

Figure 3

Cell death induced by Phytophthora cactorum RXLR effector genes (A) Pc741, (B) Pc8318, (C) Pc10890, (D) Pc16451, (E) Pc20813, (F) Pc22254, and (G) Pc22290 after transient expression in Nicotiana benthamiana leaves. The lower panel confirms cell death response after trypan blue staining. INF1, an elicitor gene of cell death from Phytophthora infestans was used as a positive control (+), and the empty vector pK7WG2 was used as a negative control (-) in each of the leaves. In addition, two previously reported cell death inducing RXLR genes (D) Pc16451 and (F) Pc22254 from P. cactorum (Chen et al., 2014) were included as positive controls. Images were taken five days after agroinfiltration of the gene constructs and after trypan blue staining of the same leaf. Pc is the abbreviated form of PC110_g from the gene identifier and is obtained from P. cactorum accession GCA_003287315.1.