Suppression of Plant Resistance Gene-Based Immunity by a Fungal Effector

1Long periods of co-evolution of plants and microorganisms have led to complex mechanisms of attack and defence, involving the innate immune system of plants and virulence factors of pathogens. The first layer of plant defence, called basal immunity, is based on recognition of conserved microbial molecules but can be suppressed by microbial virulence factors known as “effectors”. Plants respond to this suppression by employing a second layer of defence, Resistance (R) gene-based immunity, which relies on recognition of effectors. In turn, at least bacterial pathogens have found ways to manipulate or evade this second layer of defence. It is unclear to what extent this capacity exists in eukaryotic plant pathogens like oomycetes and fungi.

2Like bacteria, many plant-pathogenic fungi secrete proteins that are recognized by R-genes,. One of these fungi is Fusarium oxysporum, a common soil inhabitant. It propagates asexually and is mostly harmless. However, pathogenic and host-specific clonal lines have evolved that cause severe diseases in crops, such as banana, cotton, cucumber, melon and tomato,. Many of these diseases are caused by colonisation of the water-conducting xylem system of the roots followed by upward growth through xylem vessels, with wilting and death as a dramatic result. Strains of F. oxysporum that cause wilt of tomato plants are grouped in forma specialis (f.sp.) lycopersici. Several polymorphic resistance (R) genes have been identified in the tomato gene pool that each confer resistance against a subset of F. oxysporum f.sp. lycopersici (Fol) strains. These are I (for Immunity), I-1, I-2 and I-3. Races of Fol are named historically according to the R gene that is effective against them: the I gene and the (unlinked) I-1 gene are effective against race 1, race 2 overcomes I and I-1, but is stopped by I-2, while race 3 overcomes I, I-1 and I-2 but is blocked by I-3. Race 1 strains have been further divided into subgroups based on whether or not they are able to (partially) overcome I-2 or I-3,.

3Based on the gene-for-gene hypothesis, it is assumed that disease resistance conferred by R genes in tomato requires ‘matching’ avirulence (AVR) genes in Fol. The I gene originates from Solanum [Lycopersicon] pimpinellifolium and resides on chromosome 11,, while the I-1 gene is located on chromosome 7 in another wild relative of tomato, Solanum [Lycopersicon] pennellii. The I-2 gene has been cloned and encodes an R protein of the common NB-LRR class. The I-3 gene has not yet been cloned, but the matching AVR gene has: it encodes a small protein, Six1 (“Secreted in xylem 1”), which is secreted by Fol during colonization of the xylem system and contributes to fungal virulence. Six1 is now called Avr3 to indicate its gene-for-gene relationship with the I-3 resistance gene.

4We describe here the identification and analysis of a second avirulence factor of Fol, Avr1. Surprisingly, this protein does not only act as an avirulence factor in conjunction with the I gene, but also suppresses disease resistance mediated by I-2 and I-3.

5In an initial analysis of the xylem sap proteome of tomato plants infected with Fol race 1 using 2-D gel electrophoresis and mass spectrometry, three small secreted proteins of Fol were identified in addition to Avr3 (Six1), named Six2, Six3 and Six4, and their genes cloned. We now find that one of these, Six4, is not secreted by Fol race 2 (Fig. 1). For reasons detailed below, we now call this protein Avr1. Like the AVR3 (SIX1) gene, AVR1 is surrounded by repetitive elements (Fig. 2A). In all of the race 1 strains we examined, PCR experiments detected the presence of AVR1 and no sequence polymorphism was detected in the coding regions of seven isolates from different clonal lines (see for the list of strains; 17 of these are race 1, 23 are race 2 or 3). AVR1 was not detected in race 2 or 3 strains by PCR nor is AVR1 present in the genome sequence of the race 2 strain 4287 (Fusarium oxysporum Sequencing Project; Broad Institute of Harvard and MIT (http://www.broad.mit.edu)). Absence of AVR1 or closely related genes in the race 2 and race 3 strains used in this study was confirmed by DNA gel blot analysis (Fig. 2B, lanes 4 and 7, respectively).

6To test whether AVR1 is indeed responsible for avirulence of Fol on plants carrying the I gene, we created an AVR1 gene knock-out in a race 1 strain (Fol004) through Agrobacterium-mediated transformation (Fig. 2). For the AVR1 gene, the frequency of homologous recombination leading to gene knock-out turned out to be extremely low, with only a single knock-out mutant obtained out of ∼200 transformants (Fig. 2B, lane 2). A disease assay with this mutant (avr1Δ) confirmed that indeed deletion of AVR1 leads to breaking of I-mediated disease resistance (Fig. 3A, panel A, quantified in Fig. 3B). Re-introduction of AVR1 in the avr1Δ strain (Fig. 2B, lane 3) restored the original avirulence phenotype (results not shown). In addition, we found that disease resistance conferred by the unlinked I-1 gene in tomato also depends on recognition of Avr1, since the avr1Δ strain (but not its parental strain) is virulent on a plant line carrying I-1 (line 90E402F, results not shown). This suggests that I and I-1 express the same resistance specificity.

7To confirm that the AVR1 gene is sufficient to trigger recognition by the I gene, we transformed AVR1 to a race 2 strain (Fol007) and a race 3 strain (Fol029) that do not contain AVR1 (Fig. 2B, lanes 4–9) and are virulent on I-containing tomato lines. Ten independent transformants (six of race 2 and four of race 3) containing AVR1 were unable to cause disease on I-containing plants (Fig. 3A, panels B and C, quantified in Fig. 3B), confirming the avirulence character of AVR1. In contrast to Avr3, Avr1 is dispensable for full virulence towards plants that do not contain R genes against Fol (results not shown).

8Although all Fol strains possess an intact AVR3 gene, most race 1 strains nevertheless cause disease on plants carrying only the I-3 gene. One explanation for this is that Avr1 itself is involved in suppression of I-3 mediated disease resistance. To test this, we inoculated a plant line containing only the I-3 gene with the set of Fol strains described above. The results clearly show that Avr1 indeed has this suppressive activity: deletion of AVR1 in race 1 leads to loss of virulence towards I-3 plants (Fig. 3A, panel D, quantified in Fig. 3B), while introduction of AVR1 in race 2 or race 3 leads to gain of virulence towards I-3 plants (Fig. 3A, panels E and F, quantified in Fig. 3B). Furthermore, we discovered that Avr1 also suppresses I-2-mediated disease resistance (Fig. 3A, panels D and E, quantified in Fig. 3B). This means that the ability of some race 1 strains to cause disease on I-2 plants, as observed earlier, is likely to be caused by suppression of I-2 rather than loss of AVR2. In accordance with earlier observations using I-3 plants, we found that virulence due to suppression of I-2 and I-3 is partial compared to strains lacking the corresponding AVR gene (Fig. S1). It should be noted that not all race 1 strains are virulent on I-2 and/or I-3 plants,, even though all contain AVR1 with identical sequences (results not shown). Apparently, suppression of R gene-based immunity by Avr1 is dependent on unknown factors in the genetic background of the fungus. Since suppression works in Fol007 (race 2) and Fol029 (race 3), the genetic background in which AVR1 is effective is not restricted to race 1 strains.

9Our observation that Avr1 is not required for virulence to plants without I genes may be due to the existence of other effectors that are redundant for such an activity. Alternatively, the role of Avr1 is restricted to the suppression of I-2 and I-3-mediated disease resistance. A mechanistic explanation for the latter role could be that Avr1 interferes directly with Avr2 and Avr3. However, at least Avr3 accumulates in xylem sap and remains unaltered in the presence of Avr1,. A direct interaction between the two proteins could also not be demonstrated in vitro by pull down experiments (results not shown). Unlike bacteria, pathogenic fungi are not known to inject proteins directly into plant cells, but many are known to secrete small, frequently cysteine-rich, but otherwise unrelated proteins during colonization of plants. Avr1, like Avr3, falls within this group, the predicted mature protein having 184 residues including 6 cysteines and lacking homology to other proteins. The mode of action of most of these small secreted proteins has remained unclear. Molecular targets have been described for Avr2 and Avr4 from the leaf mold Cladosporium fulvum: Avr2 is a protease inhibitor while Avr4 binds chitin in the fungal cell wall and protects it against attack by plant chitinases. These two proteins act in the apoplast to enhance fungal virulence, but others act inside plant cells. Uptake from the apoplast by plant cells has been shown directly for ToxA, a small secreted protein that acts as a host-selective toxin. This may also occur with Avr2, since I-2 is a cytoplasmic protein. Avr1, then, may interfere with the uptake of Avr2 and Avr3. Alternatively, it may be taken up itself and interfere with I-2 and I-3 or with signal transduction processes downstream of these R proteins (Fig. 4).

10Suppression of effector-triggered (R gene-mediated) immunity has been observed in bacteria,,. In plant pathogenic fungi, suppression of avirulence by unlinked loci has been demonstrated by genetics in rust fungi. In the flax rust fungus, two dominant alleles or tightly linked genes at the I (“inhibitor”) locus suppress – sometimes partially – either one (M1) or several (M1, L1,7,8,10) R genes out of 30 against flax rust,. The flax rust inhibitor locus is not itself linked to avirulence. Here, we report the identification of a fungal avirulence factor that suppresses disease resistance conferred by two R genes.

11Interpreting this phenomenon in terms of molecular arms races between plants and their pathogens, we envisage the following scenario. During evolution of the tomato-Fol pathosystem, I-2 and I-3 have evolved to recognize, respectively, Avr2 and Avr3. Since Avr3 is required for full virulence of Fol, evasion of I-3 recognition through loss of the AVR3 gene would entail a serious fitness penalty. This explains why all Fol strains analysed so far retained AVR3,. Point mutations in AVR3 preventing recognition have not been found either. A possible explanation for this is that the I-3 protein operates in accordance with the guard model, in which not the Avr3 protein itself but the effect it has on its virulence target is recognized. In any case, Fol has (partially) regained virulence towards I-3-containing plants by acquisition of AVR1, which, as shown here, suppresses the function of I-3. Subsequently, tomato responded to this ‘invention’ with the employment of the I gene, or the unlinked I-1 gene, to specifically recognize and respond to Avr1. Apparently, I and I-1 are themselves insensitive to the suppressive effect of Avr1 (Fig. 4).

12The agricultural ‘arms race’ between Fol and tomato is different from the natural one because it is dictated by successive R gene deployment in commercial cultivars. The I gene from the wild tomato relative Solanum [Lycopersicon] pimpinellifolium was the first R gene to be introgressed into tomato cultivars to resist Fusarium wilt in the 1940s. At that time, Fol strains without Avr1 may already have been present in some locations, since I-breaking race 2 strains were quickly discovered even though major outbreaks did not occur before 1960. The I-2 gene, also from S. pimpinellifolium and directed against Avr2, was introduced in commercial cultivars in the 1960s to protect tomato against Fol race 2,. The combination of I and I-2 was effective for about two decades until the appearance of race 3 in both Australia and North America, which probably emerged from a race 2 background through selection for loss or mutation of AVR2. To combat race 3, the I-3 gene was introgressed from S. pennellii. From the results presented here, we deduce that the combination of I (or I-1) and I-3 may yield durable resistance of tomato to Fusarium wilt disease of tomato, since I-3 is directed against a virulence factor (Avr3) and I (and I-1) against the suppressor of I-3 (Avr1).

13The molecular toolbox that is now gradually filling up (Avr1, Avr3, I-2) will help us to define host targets and evolutionary bottlenecks that govern the arms race in the Fol-tomato pathosystem. It also may allow development of new strategies for breeding plants with durable resistance against fungal pathogens.

14The following tomato lines were used (Fol resistance genes between brackets): GCR161 (I), 90E402F (I-1),; 90E341F (I-2) and E779 (I-3), C32 (no I gene). The following Fol strains were used: Fol004 (race 1), Fol002 (race 2), Fol007 (race 2), Fol029 (race 3), Fol004avr1Δ (Fol004 with AVR1 deleted by gene replacement), Fol004avr1Δ+AVR1 (Fol004avr1Δ transformed with AVR1), Fol007+AVR1 (Fol007 transformed with AVR1), Fol029+AVR1 (Fol029 transformed with AVR1). See Rep et al. (2005) for a more detailed description of the wild type Fol strains.

15Proteins present in xylem sap of tomato plants infected with Fol were isolated and separated with 2-dimensional gel electrophoresis as described earlier, using for the first dimension an Immobiline DryStrip of 13 cm, pH 6–11 NL (Amersham Biosciences).

16Ten day old seedlings of tomato were inoculated with a fungal spore suspension and disease was scored after three weeks as described earlier. The outcome of the disease assays was 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).

17The AVR1 disruption construct was made by PCR amplification of AVR1 upstream and downstream sequences for homologous recombination, and their insertion in front of and behind the hygromycin resistance gene in the vector pRW2h (see below): an upstream fragment, from 714 bp to 1 bp upstream of the start codon, was cloned into pRW2h between the PacI and KpnI sites, and a downstream fragment, from 375 bp after the start codon to 537 bp downstream of the stop codon, was cloned into pRW2h between the XbaI and BssHII sites (see Fig. 2A for location of the primers). The construct for complementation was made by amplification of a AVR1 expression cassette from 714 bp upstream of the start codon to 537 bp downstream of the stop codon (Fig. 2A), which was inserted between the XbaI and StuI sites of pRW1p (see below). Transformation of these constructs to Fol was done with Agrobacterium as described earlier.

18pRW2h is a binary vector for Agrobacterium-mediated transformation of fungi. It was made through insertion of a NheI-XbaI fragment from pAN7.1, carrying the hygromycin resistance gene hph under control of the Aspergillus (Emericella) nidulans gpd promoter and trpC terminator, into the unique XbaI site of pPZP-201BK. Similarly, pRW1p was derived from pPZP-201BK through insertion of a NheI-XbaI fragment from pAN8.1 carrying the phleomycin resistance gene ble under control of the same gpd promoter and trpC terminator.

19Genomic DNA of F. oxysporum was isolated according to Raeder and Broda, digested with HindIII and BamHI, separated in a 1% agarose gel and blotted to Hybond N+ according to Sambrook et al.. The probe containing the AVR1 ORF and 3′ sequences (1402 bp, Fig. 2A) was generated by PCR and contains sequences from 72 bp upstream to 537 bp downstream of the ORF. The probe was radioactively labelled with α32P dATP using the DecaLabel™ DNA labeling kit from MBI Fermentas (Vilnius, Lithuania). Hybridization was done overnight at 65°C in 0.5M phosphate buffer pH 7.2 containing 7% SDS and 1 mM EDTA. Blots were washed at 65°C with 0.2 X SSC, 0.1% SDS. The position of sequences hybridizing to the probe were visualized by phosphoimaging (Molecular Dynamics).

20The AVR1 (SIX4) locus: AM234064

21The Avr1 (Six4) protein: CAJ84000