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, 7 (7), e1002147

Multiple Translocation of the AVR-Pita Effector Gene Among Chromosomes of the Rice Blast Fungus Magnaporthe Oryzae and Related Species

Affiliations

Multiple Translocation of the AVR-Pita Effector Gene Among Chromosomes of the Rice Blast Fungus Magnaporthe Oryzae and Related Species

Izumi Chuma et al. PLoS Pathog.

Abstract

Magnaporthe oryzae is the causal agent of rice blast disease, a devastating problem worldwide. This fungus has caused breakdown of resistance conferred by newly developed commercial cultivars. To address how the rice blast fungus adapts itself to new resistance genes so quickly, we examined chromosomal locations of AVR-Pita, a subtelomeric gene family corresponding to the Pita resistance gene, in various isolates of M. oryzae (including wheat and millet pathogens) and its related species. We found that AVR-Pita (AVR-Pita1 and AVR-Pita2) is highly variable in its genome location, occurring in chromosomes 1, 3, 4, 5, 6, 7, and supernumerary chromosomes, particularly in rice-infecting isolates. When expressed in M. oryzae, most of the AVR-Pita homologs could elicit Pita-mediated resistance, even those from non-rice isolates. AVR-Pita was flanked by a retrotransposon, which presumably contributed to its multiple translocation across the genome. On the other hand, family member AVR-Pita3, which lacks avirulence activity, was stably located on chromosome 7 in a vast majority of isolates. These results suggest that the diversification in genome location of AVR-Pita in the rice isolates is a consequence of recognition by Pita in rice. We propose a model that the multiple translocation of AVR-Pita may be associated with its frequent loss and recovery mediated by its transfer among individuals in asexual populations. This model implies that the high mobility of AVR-Pita is a key mechanism accounting for the rapid adaptation toward Pita. Dynamic adaptation of some fungal plant pathogens may be achieved by deletion and recovery of avirulence genes using a population as a unit of adaptation.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Southern blot analysis of AVR-Pita homologs in Pyricularia isolates.
Genomic DNA was digested with AclI and hybridized with the 766-bp AVR-Pita fragment (APita766). (A) RFLP types (J1, J2, J3, CH, and PO) found in Oryza isolates. (B) RFLP types (Si, Sv, Pm, D1, D2, Ce1, and Ce2) found in Setaria, Panicum, Digitaria, and Cenchrus isolates.
Figure 2
Figure 2. Linkage analyses of AVR-Pita homologs.
(A) A map of chromosome 4 constructed by using an F1 population derived from a cross between O-29J and O-30C. Segregation data of three AVR-Pita homologs (AVR-Pita-J1(O-29J), AVR-Pita-J2(O-29J), and AVR-Pita-CH(O-30C), enclosed in black rectangles) and the avirulence on cv. Yashiro-mochi (Pita carrier) determined by infection assay (AvrPita, enclosed in an open oval) were combined with those of genetic markers reported by Luo et al. . A chromosome 4 – specific marker is indicated by an asterisk. “Tell” is a telomere signal produced by Southern hybridization with a telomere repeat oligonucleotide (TTAGGG)10. (B) A map of chromosome 6 constructed by using an F1 population derived from a cross between Si-6I and T-7B. An AVR-Pita homolog found in Si-6I is enclosed in a black rectangle. Chromosome 6 – specific markers are indicated by asterisks. Markers prefixed with “Tel” are telomere signals produced by Southern hybridization with the telomere repeat probe. Letters at the end of the markers (s and t) represent parents they are derived from. For example, CH4-121H-st represents two cosegregating fragments, one from Si-6I (Setaria isolate) and one from T-7B (Triticum isolate). (C) Southern blot analysis of an F1 population derived from a cross between O-23IN and T-4B. Genomic DNAs representing each meiotic product from ten tetrads (Set41 through Set54) were digested with BamHI and hybridized with telomere repeat (upper panel) and AVR-Pita (lower panel) probes. Open arrowheads indicate restriction fragments inherited in a non-Mendelian manner.
Figure 3
Figure 3. CHEF-Southern analyses of chromosomal locations of AVR-Pita homologs in Pyricularia isolates.
(A) Representative examples of chromosomal DNAs separated by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. White arrowheads indicate chromosomal bands that hybridized to the AVR-Pita probe (APita766) in (B). Black arrowheads indicate chromosomal bands that hybridized to a chromosome 7 – specific cosmid marker, T1A11 (see Figure 4A), in (C). (B) Chromosomal bands carrying AVR-Pita homologs. The chromosomal DNAs in (A) were blotted and hybridized with the AVR-Pita probe (APita766). (C) The location of chromosome 7. The membrane in (B) was reprobed with the chromosome 7 – specific cosmid marker, T1A11.
Figure 4
Figure 4. Identification of chromosome specific markers spanning the P. oryzae chromosomes.
(A) A genetic map of a cross between P. oryzae isolates, Si-1J and T-4B. Asterisks indicate chromosome-specific markers reported by Nitta et al. . Double asterisks indicate chromosome-specific SSR markers reported by Zheng et al. . Markers prefixed with “Tel” are telomere signals produced by Southern hybridization with telomere repeats (TTAGGG)10. Letters at the end of the markers (s and t) represent parents they are derived from (s, from Si-1J; t, from T-4B). Markers in red, yellow, brown, green, light blue, blue, and violet, were used in the CHEF-Southern analysis (B) for identification of chromosomes 1, 2, 3, 4, 5, 6, and 7, respectively. Markers enclosed in rectangles were used for karyotype analysis in Figure 5A. (B) Identification of homologous chromosomes in the parental isolates (Si-1J and T-4B) on a CHEF gel. Chromosomal DNAs in the parental isolates were separated on a CHEF gel and stained with ethidium bromide (the left panel). Sizes of Schizosaccharomyces pombe chromosomes are indicated on the left of the gel photograph. The gel was then blotted and probed with the RFLP markers shown in the chromosome-specific colors in (A). Seven hybridization patterns (S1T1 through S6T5) are shown in the middle seven panels with marker names on the top as examples. Based on these Southern analyses, chromosome number(s) were assigned to each band (the right panel). The diagrammatic chromosomal bands are painted in the chromosome-specific colors used in (A). S (painted in black) indicates a supernumerary chromosome described previously .
Figure 5
Figure 5. Frequent AVR-Pita translocation occurred independently from major chromosomal translocations or duplications.
(A) A diagram of electrophoretic karyotypes of Pyricularia isolates revealed by Southern analyses. Blots of CHEF gels were hybridized with the markers enclosed in colored rectangles in Figure 4A. Chromosomal bands that hybridized exclusively to markers assigned to a single, same chromosome are painted in the color assigned to the chromosome in Figure 4. Chromosomal bands that hybridized to markers assigned to two or more, different chromosomes are divided with vertical lines and painted in the colors assigned to those chromosomes. Chromosomal bands that were smaller than the average size of chromosome 7 and did not hybridize to any chromosome-specific probes were considered to be supernumerary chromosomes and are painted in black. Asterisks indicate isolates which are deduced to have suffered from chromosomal rearrangements such as translocations or duplications. (B) A diagram of chromosomal locations of AVR-Pita homologs. Chromosomal bands that hybridized to the AVR-Pita probe (APita766) were painted with the chromosome-specific colors used in (A). The RFLP types defined in Figure 1 are shown above isolate codes. Hyphens indicate isolates carrying no AVR-Pita homologs that are detectable in the genomic Southern analysis. Shaded isolates are representatives chosen for further analyses of AVR-Pita flanks (see Figure 6). Isolates from Eleusine, Triticum, Lolium, Brachiaria, Eragrostis, and Leersia are omitted from this diagram because all isolates from these hosts are non-carriers of AVR-Pita homologs (see Figure S1).
Figure 6
Figure 6. Structures around AVR-Pita homologs in representative RFLP types.
Structures of plasmid clones (in PO, Sv, and Ce1), whole fosmid clones (in J2, J1, Si, Pm, and D1), and a partial sequence of a fosmid clone (in CH) are shown with the structure of the authentic AVR-Pita reported by Orbach et al. (PO enclosed in a rectangle). AVR-Pita homologs are depicted in red at the center. Those identified as AVR-Pita1 and AVR-Pita2 in sequence analysis (Figure 7A) were labeled with “1” and “2”, respectively. Rectangles with triangles at both ends in the same direction indicate LTR-retrotransposons. The triangles are direct, long terminal repeats (LTRs). Detached triangles indicate solo-LTRs. Rectangles with a triangle at one end indicate LINE- or SINE-like elements. Rectangles with oppositely directed small triangles at their ends indicate DNA transposons. Directions of those elements are indicated by arrows. Brown and light brown rectangles indicate intact and truncated genes, respectively, other than transposable elements. Directions of those genes are indicated by the orientation of gene names. Black ovals designated as ‘Tel’ represent telomere repeats. Dark blue lines indicate corresponding supercontigs in the M. oryzae (70–15) genome database ver.6 (http://www.broadinstitute.org/annotation/fungi/magnaporthe/). The scale is shown at the bottom of the figure. A, AclI sites; H, HindIII sites.
Figure 7
Figure 7. Molecular evidence suggesting the course of evolution of the AVR-Pita family.
(A) A Bayesian tree constructed from exon sequences of AVR-Pita homologs. The number on each branch indicates posterior probability. The tree was rooted using AVR-Pita3 as an outgroup taxon. Shaded are representative homologs whose flanks were analyzed in detail (see Figure 6). Asterisks indicate homologs used for the transformation assay (see Figure 8). The function of the homologs as an avirulence gene, which were deduced from the transformation assay and sequence analyses, is represented by + (functional) and – (nonfunctional) in the “function as AVR-Pita” column. Chromosomes carrying the homologs are shown in the “chromosome” column. “S” represents a supernumerary chromosome. The RFLP types of the homologs and structures of their flanks are depicted in the right column. See Figure 6 for legends of symbols. Cl-X, Cl-Y, and Cl-Z indicate three major clusters found in the present study. (B) Structure at the 5′ flanks of AVR-Pita homologs suggesting a horizontal transfer. (C) Structures at the 3′ flanks of AVR-Pita homologs suggesting stepwise stacking of blocks of DNA fragments. Each block is painted in a distinct color. Corresponding blocks in the diagrams (upper panel) and the nucleotide sequences (lower panel) are shown in the same color. Red boxes indicate telomere repeats. Underlines indicate nucleotide substitutions.
Figure 8
Figure 8. Functional analysis of AVR-Pita homologs with respect to avirulence activity.
(A) Pathogenicity of Oryza isolate P-2b (O-8J) and its transformants expressing various AVR-Pita homologs (Y36 through Y1) on rice cultivars, Shin2 (S2) carrying pita and Yashiromochi (YM) carrying Pita, 7 days after inoculation. See Table 1 for details of the transgenes. (B) Alignment of amino acid sequences of the AVR-Pita homologs. DQ855958 is the accession number of AVR-Pita3 used as an outgroup in Figure 7A. Functional (triggers Pita-mediated resistance) and non-functional (fails to trigger Pita-mediated resistance) homologs are shown in red and green, respectively. Amino acids shown in blue letters indicate substitutions shared by the three non-functional J1 homologs in comparison with the functional J2(O-29J) and those shared by the two non-functional PO homologs in comparison with the functional PO (4224-7-8).
Figure 9
Figure 9. CHEF-Southern analyses of chromosomal locations of AVR-Pita3 in representative isolates of P. oryzae.
(A) Chromosomal DNAs separated in the contour-clamped homogeneous electric field (CHEF). The CHEF gel was stained with ethidium bromide. Note that the left five samples did not run straight. (B) Chromosomal bands carrying AVR-Pita3. The chromosomal DNAs in (A) were blotted and hybridized with the AVR-Pita3 probe (APita3-958). (C) Identification of the chromosomes hybridizing to the AVR-Pita3 probe. Chromosomes on the gel (A) were identified by reprobing the membrane (B) with chromosome – specific markers; e.g., T1-A11 and CH5-75H for chromosome 7 and T1-G4 for chromosome 6. Chromosomal bands that hybridized to the AVR-Pita3 probe in (B) were painted with the chromosome-specific colors used in Figure 5A.

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