Multiple Evolutionary Trajectories Have Led to the Emergence of Races in Fusarium oxysporum f. sp. lycopersici.

Race 1 isolates of Fusarium oxysporum f. sp. lycopersici (FOL) are characterized by the presence of AVR1 in their genomes. The product of this gene, Avr1, triggers resistance in tomato cultivars carrying resistance gene I In FOL race 2 and race 3 isolates, AVR1 is absent, and hence they are virulent on tomato cultivars carrying I In this study, we analyzed an approximately 100-kb genomic fragment containing the AVR1 locus of FOL race 1 isolate 004 (FOL004) and compared it to the sequenced genome of FOL race 2 isolate 4287 (FOL4287). A genomic fragment of 31 kb containing AVR1 was found to be missing in FOL4287. Further analysis suggests that race 2 evolved from race 1 by deletion of this 31-kb fragment due to a recombination event between two transposable elements bordering the fragment. A worldwide collection of 71 FOL isolates representing races 1, 2, and 3, all known vegetative compatibility groups (VCGs), and five continents was subjected to PCR analysis of the AVR1 locus, including the two bordering transposable elements. Based on phylogenetic analysis using the EF1-α gene, five evolutionary lineages for FOL that correlate well with VCGs were identified. More importantly, we show that FOL races evolved in a stepwise manner within each VCG by the loss of function of avirulence genes in a number of alternative ways. Plant-pathogenic microorganisms frequently mutate to overcome disease resistance genes that have been introduced in crops. For the fungus Fusarium oxysporum f. sp. lycopersici, the causal agent of Fusarium wilt in tomato, we have identified the nature of the mutations that have led to the overcoming of the I and I-2 resistance genes in all five known clonal lineages, which include a newly discovered lineage. Five different deletion events, at least several of which are caused by recombination between transposable elements, have led to loss of AVR1 and overcoming of I Two new events affecting AVR2 that led to overcoming of I-2 have been identified. We propose a reconstruction of the evolution of races in FOL, in which the same mutations in AVR2 and AVR3 have occurred in different lineages and the FOL pathogenicity chromosome has been transferred to new lineages several times.

Biju VC ，Fokkens L ，Houterman PM ，Rep M ，Cornelissen BJC ... -  《-》

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

Inami K ，Yoshioka-Akiyama C ，Morita Y ，Yamasaki M ，Teraoka T ，Arie T ... -  《PLoS One》

A new biotype of Fusarium oxysporum f. sp. lycopersici race 2 emerged by a transposon-driven mutation of avirulence gene AVR1.

Emergence of races in Fusarium oxysporum f. sp. lycopersici (Fol) is caused by loss or mutation of at least one avirulence (AVR) gene. The product of AVR1 is a small protein (Avr1) secreted by Fol in tomato xylem sap during infection. This protein triggers Fol race 1 specific resistance (I) in tomato, indicating that AVR1 is an AVR gene. Deletion of AVR1 in race 1 resulted in the emergence of race 2, and an additional mutation in AVR2 generated race 3. Previously, we reported a new biotype of race 3, KoChi-1, in which AVR1 was truncated by a transposon Hormin, which suggested a new route to evolution of races in Fol However, to date no race 2 isolate carrying Hormin-truncated AVR1 has been reported. In this report, we describe such isolates, represented by Chiba-5, in which Hormin insertion occurred in AVR1 at a position different from that in KoChi-1. AVR1 truncation in both isolates resulted in production of defective Avr1 proteins. Chiba-5 and KoChi-1 belong to different phylogenetic clades, A1 and A2, respectively, suggesting that insertion of Hormin in AVR1 in Chiba-5 and KoChi-1 occurred as independent evolutionary events.

Kashiwa T ，Suzuki T ，Sato A ，Akai K ，Teraoka T ，Komatsu K ，Arie T ... -  《-》

Identification of I-7 expands the repertoire of genes for resistance to Fusarium wilt in tomato to three resistance gene classes.

The tomato I-3 and I-7 genes confer resistance to Fusarium oxysporum f. sp. lycopersici (Fol) race 3 and were introgressed into the cultivated tomato, Solanum lycopersicum, from the wild relative Solanum pennellii. I-3 has been identified previously on chromosome 7 and encodes an S-receptor-like kinase, but little is known about I-7. Molecular markers have been developed for the marker-assisted breeding of I-3, but none are available for I-7. We used an RNA-seq and single nucleotide polymorphism (SNP) analysis approach to map I-7 to a small introgression of S. pennellii DNA (c. 210 kb) on chromosome 8, and identified I-7 as a gene encoding a leucine-rich repeat receptor-like protein (LRR-RLP), thereby expanding the repertoire of resistance protein classes conferring resistance to Fol. Using an eds1 mutant of tomato, we showed that I-7, like many other LRR-RLPs conferring pathogen resistance in tomato, is EDS1 (Enhanced Disease Susceptibility 1) dependent. Using transgenic tomato plants carrying only the I-7 gene for Fol resistance, we found that I-7 also confers resistance to Fol races 1 and 2. Given that Fol race 1 carries Avr1, resistance to Fol race 1 indicates that I-7-mediated resistance, unlike I-2- or I-3-mediated resistance, is not suppressed by Avr1. This suggests that Avr1 is not a general suppressor of Fol resistance in tomato, leading us to hypothesize that Avr1 may be acting against an EDS1-independent pathway for resistance activation. The identification of I-7 has allowed us to develop molecular markers for marker-assisted breeding of both genes currently known to confer Fol race 3 resistance (I-3 and I-7). Given that I-7-mediated resistance is not suppressed by Avr1, I-7 may be a useful addition to I-3 in the tomato breeder's toolbox.

Gonzalez-Cendales Y ，Catanzariti AM ，Baker B ，Mcgrath DJ ，Jones DA ... -  《-》

Detection and differentiation of Fusarium oxysporum f. sp. lycopersici race 1 using loop-mediated isothermal amplification with three primer sets.

Fusarium oxysporum f. sp. lycopersici (Fol) causes tomato wilt. Based on the difference in pathogenicity towards tomato cultivars, Fol is classified into three races. In this study, a rapid method is developed for the detection and discrimination of Fol race 1 using a loop-mediated isothermal amplification (LAMP) assay with two primer sets targeting a region of the nucleotide sequence of the SIX4 gene specific for race 1 and a primer set targeting the SIX5 gene, conserved in all known Fol isolates. Upon LAMP reaction, amplification using all three primer sets was observed only when DNA of Fol race 1 was used as a template, and not when DNA of other Fol races or other fungal species was used. This method could detect 300 fg of Fol race 1 DNA, a 100-fold higher sensitivity than that obtained by conventional PCR. The method can also detect DNA extracted from soil artificially infested with Fol race 1. It is now possible to detect Fol race 1 in colonies and infected tomato stems without DNA isolation. This method is a rapid and simple tool for discrimination of Fol race 1. This study developed a loop-mediated isothermal amplification (LAMP) assay for detection and differentiation of Fusarium oxysporum f. sp. lycopersici (Fol) race 1 by using three primer sets targeting for the SIX4 and SIX5 genes. These genes are present together only in Fol race 1. This method can detect Fol race 1 in infected tomato stems without DNA extraction, affording an efficient diagnosis of Fusarium wilt on tomatoes in the field.

Ayukawa Y ，Komatsu K ，Kashiwa T ，Akai K ，Yamada M ，Teraoka T ，Arie T ... -  《-》