A genetic mechanism for emergence of races in Fusarium oxysporum f. sp. lycopersici: inactivation of avirulence gene AVR1 by transposon insertion

Abstract

Compatible/incompatible interactions between the tomato wilt fungus Fusarium oxysporum f. sp. lycopersici (FOL) and tomato Solanum lycopersicum are controlled by three avirulence genes (AVR1-3) in FOL and the corresponding resistance genes (I-I3) in tomato. The three known races (1, 2 and 3) of FOL carry AVR genes in different combinations. The current model to explain the proposed order of mutations in AVR genes is: i) FOL race 2 emerged from race 1 by losing the AVR1 and thus avoiding host resistance mediated by I (the resistance gene corresponding to AVR1), and ii) race 3 emerged when race 2 sustained a point mutation in AVR2, allowing it to evade I2-mediated resistance of the host. Here, an alternative mechanism of mutation of AVR genes was determined by analyses of a race 3 isolate, KoChi-1, that we recovered from a Japanese tomato field in 2008. Although KoChi-1 is race 3, it has an AVR1 gene that is truncated by the transposon Hormin, which belongs to the hAT family. This provides evidence that mobile genetic elements may be one of the driving forces underlying race evolution. KoChi-1 transformants carrying a wild type AVR1 gene from race 1 lost pathogenicity to cultivars carrying I, showing that the truncated KoChi-1 avr1 is not functional. These results imply that KoChi-1 is a new race 3 biotype and propose an additional path for emergence of FOL races: Race 2 emerged from race 1 by transposon-insertion into AVR1, not by deletion of the AVR1 locus; then a point mutation in race 2 AVR2 resulted in emergence of race 3.

Figure 1. Virulence of KoChi-1 and its transformants.

(A) KoChi-1 and its AVR1-complements were subjected to pathogenicity evaluation using four tomato cultivars, Ponderosa (i i2 i3), Momotaro (I i2 i3), Walter (I I2 i3) and Block (I I2 I3). The cv. Ponderosa does not have resistance to all FOL races, cv. Momotaro is resistant to FOL race 1 and susceptible to races 1 and 2, cv. Walter is susceptible to race 3 and resistant to races 1 and 2, and cv. Block is resistant to all FOL races. Inocula are as follows: KoChi-1 and its three transformants, K-B-b, K-2-11 and K-2-12; controls, race 1 MAFF 305121 (AVR1 AVR2 AVR3), race 2 JCM 12575 (– AVR2 AVR3) and race 3 Chz1-A (– avr2 AVR3). As a negative control, sterilized water was used (Mock). After three weeks of inoculation. (B) The disease severity of each individual was evaluated on external symptoms with 0∼4 scale, respectively. The external symptoms were scored as follows: 0, no wilt or yellowing; 1, lower leaves are yellowing; 2, lower and upper leaves are yellowing; 3, lower leaves are yellowing and wilt and upper leaves are yellowing; 4, all leaves are wilt and yellowing or dead. The symptoms were evaluated after three weeks of inoculation. Four plants were used in each isolate, with three replicates.

Figure 2. AVR1 in KoChi-1 genome was truncated by a transposon Hormin.

(A) Southern blot analysis to investigate the copy number of AVR1 gene. AVR1 probe was prepared using a primer set SIX4F/SIX4R (Table 3), and each gDNA was digested with restriction enzyme, EcoRV or NdeI (Fig. 2C). (B) Detection of AVR1 locus from KoChi-1 using a primer set SIX4f-F2/SIX4f-R2 (Table 3, Fig. 2C). (C) Schematic representation of AVR1 locus and AVR1 gene truncated by a transposon Hormin (avr1). The nonautonomous transposon Hormin (759 bp, shown in orange square) is inserted in the second exon of AVR1 in KoChi-1, Hormin harbors 15-bp tandem inverted repeats (TIRs, shown in blue triangle in white square) “CAGGGTTCAAATCCA” and 8-bp target site duplications (TSDs, shown with black line) “CACACCGG”. Arrows show primers. E, EcoRV site; N, NdeI site.

Figure 3. Gene expression of AVR1, avr1, AVR2 (avr2) and AVR3.

Eight days after inoculation with race 1 MAFF 305121 (AVR1 AVR2 AVR3), race 3 KoChi-1 (avr1 avr2 AVR3) and the three transformants (avr1 AVR1 avr2 AVR3); K-B-b, K-2-11 and K-2-12, total RNA was extracted from the roots of tomato (cv. Ponderosa) and investigated the transcription of genes AVR1, avr1, AVR2 (avr2), AVR3, FEM1 and Actin with the primer sets SIX4F/SIX4R, SIX4F/hornet-like2, FP962/FP963, P12-F1/P12-R1, FP157/FP158 and Actin-f/Actin-r, respectively (Table 3). FEM1 and actin are used as controls for constitutively-expressed genes in fungal and plant tissues, respectively. Sterilized water is used as a negative control.

Figure 4. Localization of avr1/AVR1, AVR2 and AVR3 on the chromosomes of KoChi-1 and other FOL isolates.

(A) Karyotype of FOL isolates by CHEF-gel electrophoresis. Electrophoresis was performed in 1.0% Sea Kem gold agarose gel with CHEF Mapper XA Pulsed Field Electrophoresis System, as following condition; 260 hours run at 8°C, 1200-4800 s switch time at 1.5 V/cm. MAFF 305121 (AVR1 AVR2 AVR3, Japan); MAFF 103036 (AVR1 AVR2 AVR3, Japan); 73 (AVR1 AVR2 AVR3, Italy); Ita3 (AVR1 AVR2 AVR3, Italy); JCM 12575 (– AVR2 AVR3, Japan); NRRL 34936 (– AVR2 AVR3, Spain); Chz1-A (– avr2 AVR3, Japan); KoChi-1 (avr1 avr2 AVR3, Japan). The chromosomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe were used as CHEF DNA size markers. (B) Southern blot analysis probed with AVR1 (upper), AVR2 (middle) and AVR3 (bottom). Probes to detect AVR1 (avr1), AVR2 (avr2) and AVR3 were prepared using primer sets SIX4F/SIX4R, SIX3-F1/SIX3R2 and P12-F2/P12-R1, respectively (Table 3).