The AvrM effector from flax rust has a structured C-terminal domain and interacts directly with the M resistance protein

Abstract

In plant immunity, recognition of pathogen effectors by plant resistance proteins leads to the activation of plant defenses and a localized cell death response. The AvrM effector from flax rust is a small secreted protein that is recognized by the M resistance protein in flax. Here, we investigate the mechanism of M-AvrM recognition and show that these two proteins directly interact in a yeast two-hybrid assay, and that this interaction correlates with the recognition specificity observed for each of the different AvrM variants. We further characterize this interaction by demonstrating that the C-terminal domain of AvrM is required for M-dependent cell death, and show that this domain also interacts with the M protein in yeast. We investigate the role of C-terminal differences among the different AvrM proteins for their involvement in this interaction and establish that M recognition is hindered by an additional 34 amino acids present at the C terminus of several AvrM variants. Structural characterization of recombinant AvrM-A protein revealed a globular C-terminal domain that dimerizes.

Fig. 1

AvrM proteins interact with the M protein in yeast. A and B, Growth of yeast strain AH109 expressing GAL4-activation domain (AD) and -binding domain (BD) fusion proteins on minimal media lacking histidine. Growth indicates expression of the HIS3 reporter gene as a result of interaction between the GAL4-AD and GAL4-BD fusion proteins. A, AD and BD proteins were expressed alone (–) or as fusions to AvrM-A (A), avrM (a), or M. B, Co-expression of the AD–M fusion protein and the BD–AvrM (A to E) or BD–avrM fusion proteins. C, Protein extracts from yeast strain AH109 expressing AD–M, AD–AvrM (A to E), or BD–avrM were analyzed by immunoblotting with anti-GAL4-AD (α-AD) or anti-cMyc (α-cMyc). Positions and sizes of protein molecular mass standards are indicated.

Fig. 2

C-terminal domain of AvrM elicits M-dependent cell death. A, Schematic diagram showing variation between AvrM-A and avrM proteins with the size of each protein as amino acid length shown at the end. The signal peptide is shaded dark and lines indicate positions of polymorphic residues; the last three variable residues are given. The position of the N-terminal 69-amino-acid (aa) deletion and the 34-aa extension of avrM are shown. N- and C-terminal truncation sites are indicated by arrows along with the amino acid number, and the size of each full-length protein in amino acids is given. B, Leaves of transgenic W38 tobacco carrying the M gene were infiltrated with Agrobacterium cultures containing T-DNA plasmids encoding AvrM-A (left side of leaf) and avrM (right side of leaf) truncations with an N-terminal hemagglutinin (HA) epitope tag. The size of the N-terminal truncation (Δ) is given and CTΔ34 indicates that the avrM truncations were made with the 34-aa C-terminal extension removed. Leaves were photographed 3 days after infiltration. C, Protein extracts from tobacco leaf tissue (W38) and leaf tissue transiently expressing HA–AvrM-A (upper) or HA–avrM (lower) truncations were analyzed by immunoblotting with anti-HA (α-HA). Positions and sizes of protein molecular mass standards are indicated.

Fig. 3

C-terminal deletions of AvrM-A prevent M-recognition. A, Leaves of transgenic W38 tobacco carrying the M gene were infiltrated with Agrobacterium cultures containing T-DNA plasmids encoding AvrM-A C-terminal truncations (CTΔ) without the signal peptide (Δ28) and with an N-terminal hemagglutinin (HA) epitope tag. Leaves were photographed 5 days after infiltration. B, Protein extracts from tobacco leaf tissue (W38) and leaf tissue transiently expressing HA–AvrM-A C-terminal truncations were analyzed by immunoblotting with anti-HA (αHA). Positions and sizes of protein molecular mass standards are indicated.

Fig. 4

Interactions of AvrM-A truncations with M protein in yeast. A, Growth of yeast strain AH109 expressing GAL4-activation domain (AD) and -binding domain (BD) fusion proteins on minimal media lacking histidine. Upper panel: strains expressing BD–AvrM-A N-terminal (Δ; left) and C-terminal (CTΔ; right) truncations with AD alone (-ve) or AD–M fusion protein. Strains expressing the reversed fusions are shown below; AD–AvrM-A truncations with BD–green fluorescent protein (-ve) or BD–M. Lower panel: strains expressing the corresponding BD–avrM truncated proteins (N-terminal [Δ] plus C-terminal [CTΔ34] to remove an additional 34-amino-acid extension) with AD alone (-ve) or AD–M. Strains expressing the reversed fusions are shown below; AD–avrM truncations with BD alone (-ve) or BD–M. B, Protein extracts from yeast strain AH109 expressing AD–AvrM-A (upper), AD–avrM, or AD–M (lower) fusion proteins were analyzed by immunoblotting with anti-hemagglutinin (α-HA). Positions and sizes of protein molecular mass standards are indicated.

Fig. 5

Structural characterization of recombinant AvrM-A protein. A, N-terminal cleavage sites in the AvrM-A protein after limited proteolysis or storage at 4°C are indicated by arrows (only residues 29 to 148 are shown): chymotrypsin (cleaves C terminal to bulky hydrophobic residues), Lys-C (cleaves C terminal to lysine), Glu-C (cleaves C terminal to glutamate), without proteases corresponds to the cleavage site when AvrM-A was stored at 4°C without proteases. B, Determination of the molecular weight of AvrM-A: molar mass distribution plot. The solid lines indicate the trace from the refractive index detector, and the dots are the weight-average molecular weights for each slice (measured every 0.5 s). AvrM-A was analyzed on a Superdex 200 10/300 column in a buffer consisting of 20 mM Tris (pH 8.0) and 300 mM NaCl.

Fig. 6

AvrM-A self-associates in planta and in yeast. A, Immunoprecipitation of AvrM and avrM from total plant protein extracts. Proteins extracted from W38 tobacco leaves expressing pairwise combinations of N-terminal tagged (yellow fluorescent protein [YFP] or hemagglutinin [HA]) AvrM-A and avrM fusion proteins were immunoprecipitated using anti-green fluorescent protein (GFP) beads and magnetic capture. Total extracted protein (input) and immunoprecipitated proteins (IPs) were analyzed by immunoblotting with anti-GFP (α-GFP; upper) and anti-HA (αHA; lower). Proteins corresponding to YFP–AvrM-A (YFP-A), HA–AvrM-A (HA–A), YFP–avrM (YFP-a), and HA–avrM (HA–a) are indicated by arrowheads on the right. Positions and sizes of protein molecular mass standards are shown. B, Growth of yeast strain AH109 expressing GAL4-activation domain (AD) and -binding domain (BD) fusion proteins on minimal media lacking histidine. Strains expressing BD–AvrM-A Δ28 with AD–AvrM-A N-terminal (Δ; upper) or C-terminal (CTΔ; lower) truncations, or AD–avrM Δ28. Strains expressing the AD–AvrM-A Δ107 with BD–AvrM-A N-terminal truncations or corresponding BD–avrM truncated protein (Δ45/CTΔ34).