Effector prediction and characterization in the oomycete pathogen Bremia lactucae reveal host-recognized WY domain proteins that lack the canonical RXLR motif

Abstract

Pathogens that infect plants and animals use a diverse arsenal of effector proteins to suppress the host immune system and promote infection. Identification of effectors in pathogen genomes is foundational to understanding mechanisms of pathogenesis, for monitoring field pathogen populations, and for breeding disease resistance. We identified candidate effectors from the lettuce downy mildew pathogen Bremia lactucae by searching the predicted proteome for the WY domain, a structural fold found in effectors that has been implicated in immune suppression as well as effector recognition by host resistance proteins. We predicted 55 WY domain containing proteins in the genome of B. lactucae and found substantial variation in both sequence and domain architecture. These candidate effectors exhibit several characteristics of pathogen effectors, including an N-terminal signal peptide, lineage specificity, and expression during infection. Unexpectedly, only a minority of B. lactucae WY effectors contain the canonical N-terminal RXLR motif, which is a conserved feature in the majority of cytoplasmic effectors reported in Phytophthora spp. Functional analysis of 21 effectors containing WY domains revealed 11 that elicited cell death on wild accessions and domesticated lettuce lines containing resistance genes, indicative of recognition of these effectors by the host immune system. Only two of the 11 recognized effectors contained the canonical RXLR motif, suggesting that there has been an evolutionary divergence in sequence motifs between genera; this has major consequences for robust effector prediction in oomycete pathogens.

EffectorO: motif-independent prediction of effectors in oomycete genomes using machine learning and lineage-specificity

“…Training sets: Positive (effectors) and negative (non-effectors) Our positive training set consisted of oomycete effectors experimentally shown to be either recognized by R genes (avirulence activity) or to have an immune-suppressing effect on the host (virulence activity) (Supplemental Table 1). Seventy-seven of these sequences were obtained from the Pathogen-Host Interaction database (PHI-base) (Urban et al 2019) and 15 effector candidates were added from B. lactucae that had avirulence (Giesbers et al 2017;Pelgrom et al 2019;Stassen et al 2013;Wood et al 2020) or virulence activity (Wood et al 2020) resulting in an initial set of 92 sequences. We then used CD-Hit at a sequence identity threshold of 70% and word length of five amino acids in order to identify and remove closely related sequences (Fu et al 2012;Li and Godzik 2006), resulting in a final positive training set of 88 unique oomycete effectors.…”

“…Two distinct alleles were obtained. Agrobacterium tumefaciens strain C58Rif+ was transformed with pEG100-BLE01 and infiltrations were performed on three- to five-week-old lettuce plants as described previously (Wood et al 2020). Leaves were scored for necrosis 4-5 days after infiltration.…”

“…Another approach to predict effectors in downy mildew species (as well as in Phytophthora ) is to search for the structurally conserved WY domain, which is present in ~25–50% of RXLR effectors in Phytophthora and downy mildew pathogens (Bos et al 2010; Dou et al 2008; He et al 2019; King et al 2014; Win et al 2012) and has been implicated in effector function (Bos et al 2010; Dou et al 2008; He et al 2019; King et al 2014). While HMM searches for the WY domain have a low false positive rate for predicting candidate effectors (Wood et al 2020), they likely have a high false negative rate, as not all RXLR effectors have WY domains and there may be bona fide effectors with novel N-terminal motifs and uncharacterized effector domains. Unlike in oomycetes, there are no widely conserved effector motifs across all fungi.…”

The hop downy mildew pathogen Pseudoperonospora humuli

“…Some RXLRs possess an additional motif in the C‐terminus involving repeats of the amino acids W, Y, and L (Haas et al, 2009; Jiang et al, 2008). Recent reports emphasize the importance of WY domain‐containing effectors that lack a canonical RXLR motif in downy mildew pathogens (Derevnina et al, 2015; Wood et al, 2020). In line with this, there were 74 such effectors in the secretome of P .…”

EffectorP 3.0: prediction of apoplastic and cytoplasmic effectors in fungi and oomycetes

“…Furthermore, the RxLR motif is not conserved outside Phytophthora species and downy mildews. The WY domain is a structural domain associated with cytoplasmic oomycete effectors from the Peronosporales that has been used to identify effector candidates with degenerate RxLR motifs (Wood et al , 2020). Similarly, degenerate RxLR motif searches have been applied in Pythium species that lack effectors with a canonical RxLR motif (Ai et al , 2020).…”