Suppression of plant resistance gene-based immunity by a fungal effector

Abstract

The innate immune system of plants consists of two layers. The first layer, called basal resistance, governs recognition of conserved microbial molecules and fends off most attempted invasions. The second layer is based on Resistance (R) genes that mediate recognition of effectors, proteins secreted by pathogens to suppress or evade basal resistance. Here, we show that a plant-pathogenic fungus secretes an effector that can both trigger and suppress R gene-based immunity. This effector, Avr1, is secreted by the xylem-invading fungus Fusarium oxysporum f.sp. lycopersici (Fol) and triggers disease resistance when the host plant, tomato, carries a matching R gene (I or I-1). At the same time, Avr1 suppresses the protective effect of two other R genes, I-2 and I-3. Based on these observations, we tentatively reconstruct the evolutionary arms race that has taken place between tomato R genes and effectors of Fol. This molecular analysis has revealed a hitherto unpredicted strategy for durable disease control based on resistance gene combinations.

Figure 1. Fol race 2 does not secrete Avr1/Six4.

Proteins present in xylem sap of susceptible tomato plants infected with race 1 strain Fol004 (left panel) or race 2 strain Fol002 (right panel) were isolated and separated with 2-dimensional gel electrophoresis. Positions of isoelectric point markers are indicated at the top; positions of molecular weight markers are indicated on the left. The arrows in the left panel point to the two spots previously shown to contain Avr1 (Six4) ; the arrows in the right panel point to the corresponding (empty) positions. The right spot in the left panel likely represents a more extensively N-terminally processed form of Avr1 .

Figure 2. The AVR1 locus, gene deletion and complementation.

A) The AVR1 open reading frame (ORF; open arrow) is interrupted by a single intron (black box) (accession AM234064). The ORF is flanked 714 bp upstream by a copy of the transposon Tfo1 (striped arrow represents the end of the transposase ORF; triangle represents the inverted repeat), 485 bp upstream by a partial miniature impala repetitive element (mimp-Δ, grey box; triangle represents inverted repeat) and downstream by a Fot5-like repetitive element (the transposase ORF ends 541 bp downstream of the AVR1 ORF and is shown as a grey arrow). The small arrows denote the primers used to construct an AVR1 disruption construct and an AVR1 expression cassette for transformation to Fol (see Materials and methods). The insertion of a hygromycine resistance (hygR) cassette to create an AVR1 knock-out mutant is shown (not drawn to scale). The position of the probe and the restriction sites used for Southern blot analysis are indicated; H: HindIII, B: BamHI. B) Southern blot confirming AVR1 disruption and ectopic insertion of AVR1. A Southern blot of genomic DNA digested with HindIII and BamHI was probed with a 1.4 kb probe encompassing the AVR1 ORF and 3′ sequences as indicated in Fig. 2A. The AVR1 locus in race 1 strain Fol004 (lane 1) is visible as a 1.25 kb HindIII band containing the ORF (AVR1) and a band of ∼5 kb containing sequences 3′ of the ORF (3′). In the race 1 avr1Δ strain (lane 2), replacement of the ORF with the disruption cassette through homologous recombination led to the expected replacement of the 1.25 HindIII band with a 1.1 kb BamHI-HindIII band containing part of the ORF and part of the disruption cassette (avr1Δ). Transformation of the AVR1 expression cassette to the avr1Δ strain (lane 3) led to reappearance of the AVR1 band. Race 2 strain Fol007 (lane 4) and race 3 strain Fol029 (lane 7) do not contain AVR1 (the AVR1 and 3′ bands are absent). Transformation of the AVR1 expression cassette to these strains (lanes 5 and 6: race 2 transformants; lanes 8 and 9: race 3 transformants) leads to appearance of the 1.25 kb HindIII AVR1 band as well as a 0.56 kb HindIII-BamHI band (3′ ectopic) that comprises sequences 3′ of the AVR1 ORF until the BamHI site at the 3′ end of the expression cassette (which is not present in the genomic locus but corresponds to the end of the probe shown in Fig. 2A). Note that in the avr1Δ strain (lane 2) the 0.56 kb band indicative of ectopic insertion is also present, indicating that this strain contains an additional copy of the disruption cassette. The additional, weaker bands are probably due to 104 bp of non-coding sequence of the Fot5-like transposon present at the 3′ end of the probe (thick line next to the grey arrow in Fig. 2A) – there are seven copies of this sequence in the latest release of the genome sequence of race 2 strain 4287 (Fusarium oxysporum Sequencing Project; Broad Institute of Harvard and MIT (http://www.broad.mit.edu). Molecular weight markers are indicated on the left (in kb).

Figure 3. Avr1 suppresses I-2 and I-3 mediated resistance.

Ten day old seedlings of tomato were inoculated with a fungal spore suspension and disease was scored after three weeks. Tomato lines carrying only a single resistance gene or no resistance gene were used to determine the effect of Avr1 on the activity of each resistance gene (see Materials and methods for description of plant lines). All lines were inoculated with the following Fol strains: race 1 (strain Fol004), race 2 (strain Fol007), race 3 (strain Fol029), race 1 avr1Δ (Fol004 with AVR1 deleted by gene replacement), race 2+AVR1 (Fol007 transformed with AVR1; similar virulence patterns were obtained with six independent transformants ) and race 3+AVR1 (Fol029 transformed with AVR1; similar virulence patterns were obtained with four independent transformants). A) Representative plants are shown three weeks after infection. Panel A shows that loss of AVR1 leads to breaking of I-mediated resistance. Panel B and C show that gain of AVR1 triggers I-mediated resistance. Panel D shows that loss of AVR1 leads to loss of virulence on I-2 and I-3-containing plant lines. Panels E and F show that gain of AVR1 by race 2 or race 3 leads to virulence on I-2 and I-3-containing plant lines. B): Quantification of disease assays. The outcomes of the disease assays depicted in (A) were quantified in two ways: 1) average plant weight above the cotyledons and 2) phenotype scoring according to a disease index ranging from zero (no disease) to four (heavily diseased or dead). Error bars indicate the 95% confidence interval of the mean. Interactions where Avr1 induces I-mediated resistance are indicated with a circle. Interactions where Avr1 suppresses I-2 or I-3 are indicated with an asterisk. N.I: not infected.

Figure 4. Schematic summary of the interactions between Fol Avr proteins and tomato resistance (I) proteins.

Arrows signify activation, lines ending in a cross bar signify suppression. Avr1 is synonymous to Six4, Avr3 is synonymous to Six1.