Mining oomycete proteomes for metalloproteases leads to identification of candidate virulence factors in Phytophthora infestans

Abstract

Pathogens deploy a wide range of pathogenicity factors, including a plethora of proteases, to modify host tissue or manipulate host defences. Metalloproteases (MPs) have been implicated in virulence in several animal and plant pathogens. Here we investigated the repertoire of MPs in 46 stramenopile species including 37 oomycetes, 5 diatoms, and 4 brown algae. Screening their complete proteomes using hidden Markov models (HMMs) trained for MP detection resulted in over 4,000 MPs, with most species having between 65 and 100 putative MPs. Classification in clans and families according to the MEROPS database showed a highly diverse MP repertoire in each species. Analyses of domain composition, orthologous groups, distribution, and abundance within the stramenopile lineage revealed a few oomycete-specific MPs and MPs potentially related to lifestyle. In-depth analyses of MPs in the plant pathogen Phytophthora infestans revealed 91 MPs, divided over 21 protein families, including 25 MPs with a predicted signal peptide or signal anchor. Expression profiling showed different patterns of MP gene expression during pre-infection and infection stages. When expressed in leaves of Nicotiana benthamiana, 12 MPs changed the sizes of lesions caused by inoculation with P. infestans; with 9 MPs the lesions were larger, suggesting a positive effect on the virulence of P. infestans, while 3 MPs had a negative effect, resulting in smaller lesions. To the best of our knowledge, this is the first systematic inventory of MPs in oomycetes and the first study pinpointing MPs as potential pathogenicity factors in Phytophthora.

Keywords: MEROPS; oomycete; peptidase; plant pathogen; secreted metalloproteases; stramenopile; virulence.

FIGURE 1

Pipeline for the identification of putative metalloproteases in stramenopiles

FIGURE 2

Metalloproteases (MPs) in stramenopiles. MP families (lower bar) were identified in 46 stramenopiles, including 37 oomycetes, and divided over various taxonomic lineages (left). Approximate numbers of MP family members in each species are shown by frequency (centre) and in bar charts (right). MP families are categorized in colour‐coded clans (top right)

FIGURE 3

Clustering of stramenopile metalloproteases (MPs) in orthogroups. MP family members (y axis) are clustered in one (one dot) or more (dots connected by horizontal lines) orthogroups. The sizes of the coloured dots correlate with the number of MP sequences per orthogroup. The first orthogroup is the largest with 285 members. For gene IDs clustered per orthogroup see Table S1

FIGURE 4

Phylogenetic tree and domain composition of M24 family members in Phytophthora infestans. Phylogram of metalloprotease subdomain sequences with bootstrap values indicated at nodes (left). Clustering based on orthogroup classification (light grey) and domain composition (dark grey) of full‐length P. infestans M24 members (right). Individual domains of M24 members are indicated

FIGURE 5

Clustering of Phytophthora infestans metalloprotease (MP) genes based on expression patterns during asexual development and in planta growth. The dendrogram on the left shows the hierarchical clustering of expression patterns. Column S marks MPs with a predicted signal peptide (dark grey) or signal anchor (light grey). Colour‐coded bars in column C indicate MEROPS clans. The heatmap depicts relative expression per gene based on stage‐wise, Z‐score transformed expression values in mycelium (MY), sporangia (SP), zoospores (ZP), germinating cysts (GC), and early, mid, and late infection stages (EI, MI, and LI, respectively). The adjacent bar plot shows mean transcripts‐per‐million mapped reads (TPM) values. Line plots on the right display the expression profiles of the eight clusters and number (n) of genes per cluster. Further details can be found in Table S2

FIGURE 6

Phytophthora infestans genes encoding putative secreted metalloproteases (MPs) and their capacity to promote or inhibit virulence. MPs are shown with a predicted signal peptide (⊳), signal anchor (◻), or mitochondrial transit peptide (❍). Numbers in grey squares refer to expression clusters (Figure 5). MPs marked with an asterisk (*) have accessory domains. Bars on the right show lesion sizes on Nicotiana benthamiana leaves transiently expressing MP genes at 5 days postinoculation. Inoculation with zoospores of P. infestans strain 14‐3‐GFP was performed one day after agroinfiltration. Bar colours indicate significantly smaller (red) or larger (green) lesions or no effect (grey) when compared to the empty vector control (EV, black; analysis of variance, p < .05). Three MPs (n.d.) were not tested in planta. Bars represent average lesion sizes from three biological repeats (n = 20). Error bars indicate SD