Novel haplotypes and networks of AVR-Pik alleles in Magnaporthe oryzae

Abstract

Background: Rice blast disease is one of the most destructive fungal disease of rice worldwide. The avirulence (AVR) genes of Magnaporthe oryzae are recognized by the cognate resistance (R) genes of rice and trigger race-specific resistance. The variation in AVR is one of the major drivers of new races. Detecting the variation in the AVR gene in isolates from a population of Magnaporthe oryzae collected from rice production fields will aid in evaluating the effectiveness of R genes in rice production areas. The Pik gene contains 5 R alleles (Pik, Pikh, Pikp, Pikm and Piks) corresponding to the AVR alleles (AVR-Pik/kh/kp/km/ks) of M. oryzae. The Pik gene specifically recognizes and prevents infections by isolates of M. oryzae that contain AVR-Pik. The molecular variation in AVR-Pik alleles of M. oryzae and Pik alleles of rice remains unclear.

Results: We studied the possible evolutionary pathways of AVR-Pik alleles by analyzing their DNA sequence variation and assaying their avirulence to the cognate Pik alleles of resistance genes under field conditions in China. The results of PCR products from genomic DNA showed that 278 of the 366 isolates of M. oryzae collected from Yunnan Province, China, carried AVR-Pik alleles. Among the isolates from six regions of Yunnan, 66.7-90.3% carried AVR-Pik alleles. Moreover, 10 AVR-Pik haplotypes encoding five novel AVR-Pik variants were identified among 201 isolates. The AVR-Pik alleles evolved to virulent from avirulent forms via stepwise base substitution. These findings demonstrate that AVR-Pik alleles are under positive selection and that mutations are responsible for defeating race-specific resistant Pik alleles in nature.

Conclusions: We demonstrated the polymorphism and distribution of AVR-Pik alleles in Yunnan Province, China. By pathogenicity assays used to detect the function of the different haplotypes of AVR-Pik, for the first time, we showed the avoidance and stepwise evolution of AVR-Pik alleles in rice production areas of Yunnan. The functional AVR-Pik possesses diversified sequence structures and is under positive selection in nature.

Keywords: AVR-Pik; Effector; Evolution; Magnaporthe oryzae.

Fig. 1

The haplotype network for the 10 AVR-Pik alleles. The original AVR-Pik allele is designated as the H01 haplotype in the network. Haplotypes are separated by mutational events. All haplotypes are displayed as circles. The size of the circles corresponds to the haplotype frequency. Haplotypes H01 to H05 are the same as AB498875, AB498876, AB498877, AB498878 and AB498879 (GenBank Accession No.) of AVR-Pik and were obtained from GenBank. Green indicates avirulence to the corresponding R gene, and yellow indicates virulence to the corresponding R gene

Fig. 2

Possible scenario for M. oryzae AVR-Pik allele-rice Pik allele interactions and coevolution. Chronological order is given on the left (time order). The AVR-Pik homolog H01 (AVR-Pik-D) was derived from an ancestral M. oryzae gene. AVR-Pik-D (H01), H07 and H09 are recognized by Pikp; thus, the altered alleles AVR-Pik-E (H05) and H08 evolved. In response to this situation, another Pik allele, Pik, evolved that can recognize five alleles, namely, AVR-Pik-D (H01), H07, H09, AVR-Pik-E (H05) and H08. Then, yet another AVR-Pik allele, H06, was derived that cannot be recognized by Pikp and Pik. Next, the rice R gene Pikm was utilized that recognizes AVR-Pik-D (H01), H07, H09, AVR-Pik-E (H05), H08 and H06. Then, two more AVR-Pik alleles, namely, AVR-Pik-A (H02) and AVR-Pik-B (H03), were derived that cannot be recognized by Pikp, Pik and Pikm. Next, the rice R gene Pikh was utilized that recognizes AVR-Pik-D (H01), H07, H09, AVR-Pik-E (H05), H08, H06, AVR-Pik-A (H02) and AVR-Pik-B (H03). Then, two other AVR-Pik alleles, namely, AVR-Pik-C (H04) and H10, evolved that cannot be recognized by any of the five Pik alleles

Fig. 3

Sliding window of positively selected sites in the AVR-Pik alleles under the M8, M8a, and M7 models. The Y-axis indicates the ratio of the rate of nonsynonymous substitutions (Ka) to the rate of synonymous substitutions (Ks) (Ka/Ks); the X-axis indicates the position of the AVR-Pik amino acids in the site. The signal region of the variant structure is purple, and the black area represents the mature protein region on the label at the top of the figure