The neighbouring genes AvrLm10A and AvrLm10B are part of a large multigene family of cooperating effector genes conserved in Dothideomycetes and Sordariomycetes

Abstract

Fungal effectors (small-secreted proteins) have long been considered as species or even subpopulation-specific. The increasing availability of high-quality fungal genomes and annotations has allowed the identification of trans-species or trans-genera families of effectors. Two avirulence effectors, AvrLm10A and AvrLm10B, of Leptosphaeria maculans, the fungus causing stem canker of oilseed rape, are members of such a large family of effectors. AvrLm10A and AvrLm10B are neighbouring genes, organized in divergent transcriptional orientation. Sequence searches within the L. maculans genome showed that AvrLm10A/AvrLm10B belong to a multigene family comprising five pairs of genes with a similar tail-to-tail organization. The two genes, in a pair, always had the same expression pattern and two expression profiles were distinguished, associated with the biotrophic colonization of cotyledons and/or petioles and stems. Of the two protein pairs further investigated, AvrLm10A_like1/AvrLm10B_like1 and AvrLm10A_like2/AvrLm10B_like2, the second one had the ability to physically interact, similarly to what was previously described for the AvrLm10A/AvrLm10B pair, and cross-interactions were also detected for two pairs. AvrLm10A homologues were identified in more than 30 Dothideomycete and Sordariomycete plant-pathogenic fungi. One of them, SIX5, is an effector from Fusarium oxysporum f. sp. lycopersici physically interacting with the avirulence effector Avr2. We found that AvrLm10A/SIX5 homologues were associated with at least eight distinct putative effector families, suggesting that AvrLm10A/SIX5 is able to cooperate with different effectors. These results point to a general role of the AvrLm10A/SIX5 proteins as "cooperating proteins", able to interact with diverse families of effectors whose encoding gene is co-regulated with the neighbouring AvrLm10A homologue.

Keywords: Brassica napus; Fusarium oxysporum; Leptosphaeria maculans; avirulence; effector family; pathogenic fungi; resistance.

FIGURE 1

Presence of the AvrLm10 family in natural populations of Leptosphaeria maculans ‘brassicae’. Presence of the genes was evaluated by PCR using 5′ and 3′ untranslated region (UTR)‐specific primers (see Table S1). Absence of a gene amplification is represented by a white box.

FIGURE 2

Expression of the AvrLm10 family during oilseed rape infection by Leptosphaeria maculans ‘brassicae’. (a) Expression pattern of the AvrLm10 gene family using RNA‐seq data generated by Gay et al. (2021) and normalized by the total number of sequences per condition (count per million, CPM). Each data point is the average of two independent biological replicates. (b) Expression pattern of the AvrLm10 gene family analysed by reverse transcription‐quantitative PCR. Gene expression levels are relative to EF1α, a constitutively expressed gene, according to Muller et al. (2002). Each data point is the average of two biological replicates and two technical replicates. Standard error of the mean normalized expression level is indicated by error bars. RNA extractions were performed on cotyledons, petioles, and stems of oilseed rape (Darmor‐bzh) inoculated under controlled conditions with the reference isolate v23.1.2 and recovered at different dates (days postinoculation, DPI) on cotyledons (C), petioles (P), and stems (S).

FIGURE 3

AvrLm10A_like1, AvrLm10B_like1, AvrLm10A_like2, and AvrLm10B_like2 co‐localize in Nicotiana benthamiana cells, but only AvrLm10A_like2 and AvrLm10B_like2 physically interact. (a) Single‐plane confocal images of N. benthamiana epidermal leaf cells expressing AvrLm10A_like1‐GFP, AvrLm10B_like1‐RFP, AvrLm10A_like2‐GFP, and AvrLm10B_like2‐RFP at 48 h postinfiltration of Agrobacterium tumefaciens. (b) Proteins were extracted 48 h after infiltration and analysed by immunoblotting with anti‐RFP (α‐RFP) antibodies (Input). Immunoprecipitation was performed with anti‐RFP beads (IP RFP) and analysed by immunoblotting with anti‐RFP antibodies to detect AvrLm10B_like1‐RFP, AvrLm10B_like2‐RFP, and RFP, and with anti‐GFP (α‐GFP) antibodies for the detection of co‐immunoprecipitated proteins.

FIGURE 4

Eight different families of neighbouring genes cluster in the phylogenetic tree of AvrLm10A/SIX5 homologues. Maximum‐likelihood phylogeny of AvrLm10A/SIX5 homologues listed in Table S5. The shape of terminal nodes indicates whether the protein is from a Sordariomycete (circle) or Dothideomycete (square) or other (no shape), nodes are coloured according to species and labelled with the protein identifier. Putative duplications are indicated with red stars. One tandem duplication is indicated with a “T”: all Fusarium genes under that node are either adjacent to each other or at the end of separate contigs. The different families of neighbouring genes are indicated with coloured squares at the right side of the tree, together with species and strain names. The tree is divided into three main clades according to similarities in position of cysteines (Figure 5).

FIGURE 5

Sequence logos of three different subfamilies of AvrLm10A/SIX5 homologues. Sequence logos of amino acid sequences of the 25 proteins that belong to Clade I (a), of the 10 proteins that belong to Clade II (b), and of the 36 proteins that belong to Clade III (c) according to Figure 4. All sequence logos are based on position 60 to 201 in the multiple sequence alignment of AvrLm10A/SIX5 homologues and thus exclude the signal peptide. Conserved motifs in all clades are indicated at the bottom.