Insertion of transposable elements in AVR-Pib of Magnaporthe oryzae

doi:10.21203/rs.3.rs-1440283/v1

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Insertion of transposable elements in AVR-Pib of Magnaporthe oryzae

https://doi.org/10.21203/rs.3.rs-1440283/v1

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Background: Rice blast is a very serious disease caused by Magnaporthe oryzae. The avirulence (AVR) genes of rice blast are perceived by the corresponding rice-blast resistance (R) genes and prompt specific resistance. A mutantion in AVR is a major force for new virulence. Exploring mutations in AVR among M. oryzae isolates from rice-production fields could aid assessment of the efficacy and durability of R genes. We studied the probable molecular-evolutionary patterns of AVR-Pib alleles by assaying their DNA-sequence diversification and examining their avirulence to the corresponding Pib resistance gene under natural conditions in China.

Results: PCRs detected results from M. oryzae genomic DNA revealed that 162 out of 366 isolates collected from Yunnan Province contained AVR-Pib alleles. Among them, 36.1–73.3% isolates from six different rice-production areas of Yunnan contained AVR-Pib alleles. Furthermore, 36 (28.6%) out of 126 isolates had a transposable element (TE) insertion in AVR-Pib, which resulted in altered virulence. The TE insertion was identified in isolates from rice rather than from Musa nana Lour. Twelve AVR-Pib haplotypes encoding three novel AVR-Pib variants were identified among the remaining 90 isolates. AVR-Pib alleles evolved to virulent forms from avirulent forms by base substitution and TE insertion of Pot2 and Pot3 in the 5′ untranslated region of AVR-Pib.

Conclusions: Our results revealed that AVR-Pib alleles suffered neutral evolution and divergences that led to overcome specific resistant Pib alleles under field conditions. The TE insertion in AVR-Pib was selected and adapted to rice and other Gramineae species.

Magnaporthe oryzae

Rice blast

AVR-Pib

Evolution

In the coevolution of plants and pathogens, the latter can adapt to the host and environment, and selection is the major evolutionary force. Up to now, the “arms race” and “trench warfare” hypotheses of coevolution between host resistance (R) genes and pathogen avirulence (AVR) genes have been proposed. In the principal hypothesis of the arms-race, the mutation of R genes and AVR genes is derived by directional selection. Contrarily, it is derived by undirectional selection in the trench-warfare hypothesis.

Rice blast is one of the most serious diseases in rice worldwide, which caused by the fungus Magnaporthe oryzae. Application of rice varieties with multiple resistant genes is the most important method governing the disease by an economical, environmentally and friendly manner. Up to now, more than 26 rice blast R genes have been cloned in rice: Pita, PiCO39, Pish, Pi1, Pik, Pikp, Pikh/Pi54, Pikm, Pb1, Pid3, Pia, Pib, Pid2, Pit, Pizt, Pi2, Pi5, Pi9, pi21, Pi25, Pi36, Pi37, Pi56, Pi63 (www.ricedata.cn/gene/gene_pi.htm), Pi64 [1] and Pigm [2].

Based on the gene-for-gene theory, rice R gene(s) can discern the corresponding AVR of M. oryzae and trigger the defense response to prevent invasion. So far, 12 AVR genes have been cloned in M. oryzae: AVR-Pib [3], AVR-Pi54 [4], AVR-Pi9 [5], AVR-Pia [6], AVR-Pik/km/kp [6], AVR-Pii [6], AVR-Pizt [7], ACE1 [8], AVR1-CO39 [9], AVR-Pita [10], PWL1 [11], and PWL2 [12]. The AVR-Pib gene of M. oryzae predicts the resistance efficacy of the rice R gene Pib. AVR-Pib encodes a putative secreted protein with 75 amino acids. AVR-Pib is perceived by the host as a Pib resistance protein and prompts the innate immune response [3]. Transposable element (TE) insertion, segmental deletion, absence and point mutations have been identified in AVR-Pib in Chinese rice-blast isolates, which has resulted in loss of avirulence [3]. Only TE insertion has been observed in AVR-Pib in 248 M. oryzae isolates from the Philippines [13].

The rice resistance gene of Pib is located on the long arm of chromosome 2 [14–15]. The cDNA length of the Pib gene contains 306 bp of 5' untranslated regions (UTRs), 3753 bp of open reading frames (ORFs) (containing 3 exons), and 229 bp of 3' UTRs, and encodes a NBS-LRR protein with 1251 amino acids [16]. In China, 16 out of 204 (7.8%) varieties have been detected hoarding the Pib gene in a mini-core collection of Chinese rice germplasm using their functional markers [17]. Also, 11 varieties have been identified with the Pib gene among 58 leading rice cultivars or hybrid rice parents in China using functional DNA markers [18]. Pi-b has been detected in 33 landraces among 176 landraces (18.8%) from Yunnan Province in China [19]. Pib gene homologs (87-bp deletion in exon 1 of Pib, leading to loss of the resistance function of Pib) have been identified in Yunnan Yuanjiang type of common wild rice (Oryza rufipogon Griff) [20]. In the Philippines, 32 out of 52 commercial rice varieties have been shown to contain Pib as detected by polymerase chain reaction (PCR) primers specific to Pib [13]. AVR-Pib has been detected in 463 among 621 (74.6%) isolates of M. oryzae collected from Jiangsu Province in China [21], whereas TE insertion was found in the 5′ UTR of AVR-Pib [3, 13].

The effectiveness of resistance of Pib has been examined in different rice-production provinces in China. Pib exerts a high level of resistance to M. oryzae from Heilongjiang Province, and can be applied as a parent for resistance breeding in Heilongjiang Province [22]. Pib is moderately resistant in Fujian Province [23], but Pib exhibits low resistance in Guangdong, Sichuan and Guizhou Provinces [24–25]. Different resistance spectra of Pib were detected in 282 blast isolates collected from Indica rice- and Japonica rice-production regions in Yunnan Province [26]. Those results showed that the Pib gene exhibits different resistance to Chinese rice blast from different rice-growing regions. There were 42 out of 54 varieties containing Pib in Chinese elite hybrid rice varieties, two haplotypes of Pib were identified, and 9 different haplotypes of AVR-Pib were found among 27 M. oryzae [27]. The reactions of differential isolates on 54 rice varieties between the frequency of AVR-Pib haplotypes in the differential isolates showed a good correlation [27]. Those showed that the Pib gene widly distributed in rice varieties in China, and the adapt variation of AVR-Pib of M. oryzae are occurred.

Further clarification of the diversification and evolution of the AVR gene is useful for prediction of the effectiveness and durability of R genes for resistance breeding. Here, we wished to: (i) detect the diversification of nucleotide sequences of AVR-Pib alleles of M. oryzae under field conditions; (ii) determine the virulence function of AVR-Pib variations against the Pib gene; (iii) reveal the diversification and molecular-evolutionary principles of AVR-Pib alleles in M. oryzae in Yunnan Province.

Effectiveness of the Pib gene and frequency of AVR-Pib alleles

The efficacy of the Pib gene was examined by pathogenicity assays. A total of 223 of the 366 M. oryzae isolates (4000 isolates were collected from six rice growing regions from 1997 to 2012 in Yunnan Province, and total of 366 isolates are selected from six rice growing regions as representative isolates.) tested were avirulent to the Pib gene-containing rice monogenic line IRBLb-B (Table 1). The percentage of avirulent isolates to Pib was 60.9%, whereas the remaining 143 isolates were virulent to the Pib gene (Table 1). The percentage of avirulent isolate was 100, 75.9, 75.0, 57.6, 53.6, and 48.2% in northwestern, central, northeastern, southeastern, southwestern, western Yunnan Province, respectively. These results suggested that Pib loci had different efficacy against blast infections in most rice-growing regions of Yunnan Province. Among 366 isolates, three genotypes (L1 with 1231bp, L2 with 3100bp, L3 with both of 1231bp and 3100bp) of AVR-Pib alleles in 162 isolates were amplified by AVR-Pib-specific primers (AVR-Pib F1/AVR-Pib R1) (Table 1; Additional file 4: Fig. S1), and the average percentage of the AVR-Pib allele was 44.3%. The highest percentage of amplification of AVR-Pib was 73.3% in the rice-blast isolates collected from northwestern Yunnan Province, whereas the lowest percentage was 36.1% from northeastern Yunnan Province (Table 1). The percentage of AVR-Pib was 46.3, 36.1, 73.3, 51.5, 67.9, and 39.0% in central, northeastern, northwestern, southeastern, southwestern and western Yunnan Province, respectively. The percentage of AVR-Pib was 47.0 and 42.4% in Xian/Indica (XI) rice- and Geng/Japonica (GJ) rice-production areas in Yunnan. The genotype of L1, L2 and L3 alleles of AVR-Pib was detected in 104, 53 and 5 isolates, with percentages of 28.4%, 14.5% and 1.3%, respectively (Table 1). The genotype of L1, L2 and L3 alleles of AVR-Pib was detected in the XI rice-production area, whereas L3 was absent in the GJ rice-production area (Table 1). These results suggested that the genome structure of AVR-Pib loci was complicated considerably in M. oryzae.

Table 1

Frequency of *AVR-Pib* genotypes and avirulent isolates of *M. oryzae* collected from Yunnan, China to IRBLb-B
Locations	No. of isolates	PCR detection				Pathogenicity assay^b
		Genotype and No. of isolates and frequency (%)				No. of avirulence isolates and Frequency (%)
		L1	L2	L3	Total isolates and frequency (%)	No. of avirulence isolates and Frequency (%)
Central	54	22 (40.7)	3 (5.6))	0	25 (46.3)^B	41 (75.9)^AB
Northeastern	72	23 (31.9)	3 (4.2)	0	26 (36.1)^B	54 (75.0)^B
Northwestern	15	11 (73.3)	0	0	11 (73.3)^A	15 (100)^A
Southeastern	33	10 (30.3)	6 (18.2)	1 (3.0)	17 (51.5)^B	19 (57.6)^C
Southwestern	28	9 (32.1)	8 (28.6)	2 (7.1)	19 (67.9^A	15 (53.6)^C
Western	164	29 (17.7)	33 (20.1)	2 (1.2)	64 (39.0)^B	79 (48.2)^C
Total	366	104 (28.4)	53 (14.5)	5 (1.4)	162 (44.3)	223 (60.9)
XI	149	31 (20.8)	34 (22.8)	5 (3.4)	70 (47.0)^*	69 (46.3)^**
GJ	217	73 (33.6)	19 (8.8)	0	92 (42.4)^*	154 (71.0)^**
Total	366	104 (28.4)	53 (14.5)	5 (1.4)	162 (44.3)	223 (60.9)
^a L1 indicates the AVR-Pib genotype with the expected size (1231bp), L2 and L3 indicates the AVR-Pib genotype with TE insertion (L2 with 3100bp, L3 with both of 1231bp and 3100bp). The superscript of A and B indicates the significant difference at 0.01 level, and * indicates non significant.
^b Indicates pathogenicity assay on monogenic line IRBLb-B containing Pib. XI and GJ indicates Xian/Indica and Geng/Japonic, respectively. The superscript of A, B and C indicates the significant difference at 0.01 level, ** indicates significant difference at 0.01 level.

Virulence function of AVR-Pib variations against the Pib gene

Twelve AVR-Pib haplotypes (H01 to H12) (Table 2), excluding the original AVR-Pib allele (GenBank accession number, KM887844), were detected on the nucleotide sequence assemblies of 90 isolates of L1 alleles containing a 719-bp 5′-region, 225-bp coding DNA sequence (CDS), and 302-bp 3′-region of AVR-Pib (Table 2; Additional file 5: Fig. S2). Also, insertion of Pot2 (at position -275) and Pot3 (at position -240) was identified based on the DNA sequence assemblies of six and 30 isolates (Table 2; Fig. 1), respectively, and the amplicon size difference between L1 and L2 (Additional file 4: Fig. S1). The 12 novel AVR-Pib haplotypes (H01-H12) were identified compared to previous published alleles [3, 27]. Alignment of DNA sequence assemblies of the AVR-Pib gene from 90 isolates revealed 18 mutation sites, including six mutant sites in the CDS region which weren’t in the signal-peptide region (Table 2; Additional file 5 and 6: Fig. S2 and S3). Six mutant sites in the CDS region led to changes in amino acids (Table 3). The CDS sequence assemblies of the AVR-Pib allele among the 126 isolates (including L1, L2 and L3) were predicted to produce four AVR-Pib proteins (Table 3). Among them, amino-acid variations were predicted to occur at six positions (Table 3). Amino-acid variations at F54L in H05 and H06, E46V, F53S, and F54V in H07 were found, these isolates of the corresponding haplotypes were avirulent on the monogenic line IRBLb-B (with Pib), whileas, the amino-acid variations at F47L, I49T, and R50G were found in one isolate with H08, which was virulent on the monogenic line IRBLb-B (with Pib) (Table 3), the amino-acid variations at F47, I49, and R50 in H01, H02, H03, H04, H05, H06, H07, H09, H10, and H12, and these isolates were avirulent on the monogenic line IRBLb-B (with Pib), whileas the amino-acid variations at 47L, 49T, and 50G in H08, and the isolate was virulent on IRBLB-b (Table 3). This finding suggested that the amino acids F47, I49, and R50 were crucial for the avirulence function of AVR-Pib.

Table 2

Haplotypes of *AVR-Pib* loci in *M. oryza*e field populations of Yunnan, China
Haplotype	No. of isolates	% of total	Variant locus^a
			5' UTR								CDS regions						3' UTR
			-338	Between -325 and -326	Between -239 and -240	Between -216 and -217	-192	-175	Between -210 and -211	-93	137	141	146	148	158	160	+70	+154	+218	+232
KM887844			T	-	-	-	C	C	-	T	A	T	T	C	A	T	C	G	A	C
H01	33	26.2	.	.	.	C	.	.	.	.	.	.	.	.	.	.	.	.	.	.
H02	4	3.2	.	.	.	C	.	.	.	A	.	.	.	.	.	.	.	T	.	.
H03	4	3.2	.	.	.	C	T	.	.	.	.	.	.	.	.	.	.	.	.	.
H04	1	0.8	.	ACTTA	.	C	.	.	.	.	.	.	.	.	.	.	T	.	.	.
H05	8	6.3	C	.	.	C	.	.	.	.	.	.	.	.	.	C	.	.	.	.
H06	1	0.8	C	.	ACGTTA	C	.	.	.	.	.	.	.	.	.	C	.	.	.	.
H07	3	2.4	.	.	.	C	.	T	.	.	T	.	.	.	C	G	.	.	.	A
H08	1	0.8	.	.	.	C	.	.	ACA	.	.	A	C	G	.	.	.	.	.	.
H09	13	10.3	.	ACTTA	.	C	.	.	.	.	.	.	.	.	.	.	.	.	.	.
H10	10	7.9	.	AGTTA	.	C	.	.	.	.	.	.	.	.	.	.	.	.	.	.
H11	2	1.6	.	ATTA	.	C	.	.	.	.	.	.	.	.	.	.	.	.	.	.
H12	10	7.9	.	.	.	C	.	.	ACA	.	.	.	.	.	.	.	.	.	.	.
Pot2	6	4.8	-275 insert Pot2		.	C	.	.	.	.	.	.	.	.	.	.	.	.	.	.
Pot3 rev-A	1	0.8	-240 insert Pot3		.	C	.	.	.	.	.	.	.	.	.	.	.	.	.	.
Pot3 rev-B	2	1.6	-240 insert Pot3		.	C	.	.	.	.	.	.	.	.	.	C	.	.	.	.
Pot3-A	24	19.1	-240 insert Pot3		.	C	.	.	.	.	.	.	.	.	.	.	.	.	.	.
Pot3-B	2	1.6	-240 insert Pot3		.	C	.	.	.	.	.	.	.	.	.	C	.	.	.	.
Pot3-C	1	0.8	-240 insert Pot3		.	C	.	.	.	.	.	.	.	.	.	.	.	.	C	.
^a . Indicates the same with KM887844 (GenBank Accession No.). The KM887844 of AVR-Pib was obtained from GenBank. rev: indicates reverse insertion of Pot3 in AVR-Pib.

Table 3

Variation of the *AVR-Pib* loci proteins in *M. oryza*e populations of Yunnan, China
Haplotype	No. of isolates	% of total	Variant locus^a						Disease reaction^b
Haplotype	No. of isolates	% of total	46	47	49	50	53	54
KM887844			E	F	I	R	Y	F
H01	33	26.2	.	.	.	.	.	.	24R+5M+4?
H02	4	3.2	.	.	.	.	.	.	3R+1M
H03	4	3.2	.	.	.	.	.	.	4R
H04	1	0.8	.	.	.	.	.	.	1R
H05	8	6.3	.	.	.	.	.	L	7R+1?
H06	1	0.8	.	.	.	.	.	L	1R
H07	3	2.4	V	.	.	.	S	V	3R
H08	1	0.8	.	L	T	G	.	.	1S
H09	13	10.3	.	.	.	.	.	.	11R+2M
H10	10	7.9	.	.	.	.	.	.	9R+1M
H11	2	1.6	.	.	.	.	.	.	2S
H12	10	7.9	.	.	.	.	.	.	9R+1M
Pot2	6	4.8	.	.	.	.	.	.	7S
Pot3 rev-A^c	1	0.8	.	.	.	.	.	.	1S
Pot3 rev-B	2	1.6	.	.	.	.	.	L	2S
Pot3-A	24	19.1	.	.	.	.	.	.	22S+2M
Pot3-B	2	1.6	.	.	.	.	.	.	2S
Pot3-C	1	0.8	.	.	.	.	.	L	1S
^a. Indicates the same with KM887844.
^b Indicates pathogenicity assay on the monogenic lines IRBLb-B containing the resistant gene of Pib. R, M and S indicate disease reaction was resistant, moderate resistant and susceptible, respectively; Ex. 24R indicated 24 isolates were avirulent to IRBLb-B; and ? indicates unknown.
^c rev: indicates reverse insertion of Pot3 in AVR-Pib.

The different haplotypes of H01, H02, H03, H04, H09, H10, H11 and H12 had no change on amino acids sequence (Table 3). The three-dimensional protein structures built by homology modeling (SWISS-MODEL; https://swissmodel.expasy.org/) showed the different protein structures of these four AVR-Pib variants (Additional file 7: Fig. S4). Isolates of H11 with insertion of ATTA in the 5′ UTR may change AVR-Pib expression and cause loss of the avirulent function (Tables 2 and 3; Additional file 6: Fig. S3). Isolates of H01 (amino acids same as that with a GenBank accession number of KM887844), H02, H03, H04, H05, H06, H07, H09, H10 and H12 haplotypes harbored AVR-Pib because these isolates were avirulent to the Pib-containing monogenic line IRBLb-B (Table 3). Isolate of H08 defeated the resistance of Pib because this isolate was virulent to the Pib-containing monogenic line IRBLb-B (Table 3). Furthermore, Pot2 and Pot3 inserted in the 5′ UTR of the Pib gene were identified in six isolates and 30 isolates (Table 3; Fig. 1), respectively, and these isolates were virulent to the Pib-containing monogenic line IRBLb-B (Table 3). These findings suggested that insertion of TEs (Pot2 and Pot3) and small segments of the nucleotide in the promoter region, and nuclear substitution in the ORF region, resulted in variation of AVR-Pib from avirulence to virulence, and that the diverse mutations of the AVR-Pib allele of M. oryzae were involved.

Haplotype diversity of AVR-Pib of M. oryzae

Among 12 AVR-Pib haplotypes, none were identical to the original AVR-Pib (GenBank accession number, KM887844) (Table 2). Eight haplotypes, as well as Pot2 and Pot3 insertion were detected in 50 M. oryzae isolates from western Yunnan Province (Table 4). Five haplotypes, as well as Pot3 and Pot3 reverse-insertion were identified in 23 M. oryzae isolates from central Yunnan Province. Three haplotypes, as well as Pot2 and Pot3 reverse-insertion were identified in 15 isolates of M. oryzae from northeastern Yunnan Province. Three haplotypes and Pot2 insertion were detected in 19 isolates from northeastern Yunnan Province. Three haplotypes and Pot3 insertion were identified in six M. oryzae isolates from southeastern Yunnan Province. Three haplotypes were identified in 13 isolates of M. oryzae from northwestern Yunnan Province (Table 4). Eleven and nine haplotypes were detected in GJ rice- and XI rice-production areas, and the diversity index (DI) of haplotypes was 0.84 and 0.79 for these areas, respectively. The DI of AVR-Pib was 0.72, 0.71, 0.70, 0.63, 0.59, and 0.54 for southeastern, central, western, southwestern, northeastern, and northwestern Yunnan Province, respectively (Table 4). In brief, the DI of AVR-Pib alleles in Yunnan Province was in the order southeastern > central > western > southwestern > northeastern > northwestern. The DI of AVR-Pib alleles in the GJ-rice production area was higher than that of in the XI rice-production area. These results indicate that the genetic divergence of AVR-Pib of M. oryzae in each rice growing region was occurred under fields condition.

Table 4

Haplotype distribution of *AVR-Pib* in different Yunnan rice growing regions
Haplotype	No. isolates	Percent (%)	Regions						Production^c
Haplotype	No. isolates	Percent (%)	Central	Northeastern	Northwestern	Southwestern	Southeastern	Western	XI	GJ
H01	33	26.2	11(47.8)^a	9(60.0)	2(15.4)	10(52.6)	0	1(2.0)	10(20.8)	23(29.5)
H02	4	3.2	0	0	0	4(21.1)	0	0	4(8.3)	0
H03	4	3.2	4(17.4)	0	0	0	0	0	0	4(5.1)
H04	1	0.8	0	0	0	0	0	1(2.0)	0	1(1.3)
H05	8	6.3	1(4.3)	1(6.7)	3(23.1)	0	2(33.3)	1(2.0)	2(4.2)	6(7.7)
H06	1	0.8	0	0	0	0	1(16.7)	0	1(2.1)	0
H07	3	2.4	0	0	0	0	0	3(6.0)	0	3(3.8)
H08	1	0.8	0	0	0	0	0	1(2.0)	1(2.1)	0
H09	13	10.3	2(8.7)	0	0	0	0	11(22.0)	0	13(16.7)
H10	10	7.9	2(8.7)	0	8(61.5)	0	0	0	0	10(12.8)
H11	2	1.6	0	0	0	0	1(16.7)	1(2.0)	1(2.1)	1(1.3)
H12	10	7.9	0	3(20.0)	0	1(5.3)	0	6(12.0)	6(12.5)	4(5.1)
Pot2	6	4.8	0	1(6.7)	0	4(21.1)	0	1(2.0)	6(12.5)	0
Pot3 rev	3	2.4	2(8.7)	1(6.7)	0	0	0	0	0	3(3.8)
Pot3	27	21.4	1(4.3)	0	0	0	2(33.3)	24(48.0)	17	10(12.8)
Total	126	100	23	15	13	19	6	50	48	78
No. of haplotypes			7	5	3	4	4	10	9	11
Index of diversity^b			0.71	0.59	0.54	0.63	0.72	0.70	0.79	0.84
^a Number and frequency (in bracket) of isolates of each haplotype.
^b Diversity index was calculated as the frequency of haplotypes types in the M. oryzae population following Fontaine’s method [43]. Diversity index = (1-∑ⁿ_i=1p_i²) (where pi is the frequency of the haplotype i in a population).
^c XI and GJ indicates Xian/Indica and Geng/Japonic, respectively.

Eighteen nucleotide variable sites in AVR-Pib alleles were identified (Table 2; Additional file 5 and 6: Figs. S2 and S3). A haplotype network based on sequence variations of 90 isolates of L1 alleles was developed (Fig. 2). Four main lineage branches (A to D) of AVR-Pib were divided among 90 field isolates (Fig. 2), and different evolution of AVR-Pib among them was noted. Isolates of B and D lineage branches of AVR-Pib were avirulent to IRBLb-B (with Pib) (Fig. 2; Table 3). Isolates of H11 of the A-evolved branch and H08 of the C-evolved branch were virulent to the rice blast-resistant gene Pib, respectively (Fig. 2; Table 3). These data suggested that the A and C branches of AVR-Pib had evolved to virulence from avirulent origins via base substitution and insertion, and evaded the recognition of the rice blast-resistance gene Pib in field isolates. The virulence of H08 and H11 was identified in southeastern and western Yunnan Province (Table 4). Also, TE insertion in rice samples in all regions except northwestern Yunnan Province (Table 4) suggested that virulent evolution of AVR-Pib occurred in most rice-production areas of Yunnan Province.

Selection pressure on AVR-Pib in M. oryzae

The natural-selection pressure on AVR-Pib was calculated by Tajima's neutrality test on 126 AVR-Pib CDS sequences: the Tajima's D value was not significantly different from zero (D=−1.61687; 0.10>P>0.05) (Additional file 1: Table S1). This result suggested that AVR-Pib may suffer neutral selection and evolving neutrally in the population of M. oryzae. Furthermore, the results of three positive-selection models kept higher similatity (Additional file 8: Fig. S5). The “sliding window” under M8, M8a, and M7 models showed values of Ka/Ks (Ka, rate of nonsynonymous substitutions; Ks, rate of synonymous substitutions) across all 74 amino acids (Additional file 8: Fig. S5). The Ka/Ks value of all sites was >1 under M8 and M8a model, and the value was 1 under M7 model for entire residues, those results implying that these sites may have suffered neutral selection. These findings suggest that the AVR-Pib maybe under neutral evolution.

Adaption of TE insertion in AVR-Pib

We wished to confirm the host (rice and non rice) selection pressure on TE insertion in AVR-Pib. A total of 27 isolates from O. rufipogon (with Pib homologs) [20], Digitaria sanguinalis, Eleusine indica, E. coracana, and Musa nana Lour, which were stored in our lab, and 5 isolates genome sequence from Lolium perenne Linn (2 isolates), Setaria viridis (Linn.) Beauv. (1 isolate), and Triticum aestivum Linn (2 isolates) from Genbank were selected and analyzed (Additional file 2: Table S2). The AVR-Pib allele was not detected in the isolate from D. sanguinalis, M. nana Lour, Lolium perenne Linn, and Setaria viridis (Linn.) Beauv. (Additional file 2: Table S2). Only the L1 genotypes (with the expected size) of AVR-Pib were detected in isolates from E. indica, E. coracana, and Triticum aestivum Linn, suggesting that these isolates did not have a TE insertion in the AVR-Pib allele. Three genotypes of L1, L2 (with TE insertion), and L3 (with TE insertion) of AVR-Pib were detected in 18 isolates from the Pib homolog-containing O. rufipogon, and the isolate of YN441 (with the H9 haplotype of AVR-Pib and identical with the original haplotype of KM887844) was virulent to O. rufipogon (Fig. 3). These findings showed that the diversification of AVR-Pib of M. oryzae was dependent upon Pib homolog-containing O. rufipogon, and that variation in TE insertion in AVR-Pib could be selected and adapted to rice and other Gramineae species.

Phylogeny of Pib allele partial to CDS regions

Fifty-seven sequences of Pib were obtained from GenBank (Additional file 3: Table S3). Eleven of them were from five wild-rice species (seven from O. rufipogon, one from O. meyeriana, one from O. officinalis, one from O. longistaminata, one from O. nivara), and 46 accessions from O. sativa, including the original Pib (GenBank accession number, AB013448.1) (Additional file 3: Table S3). These sequences were aligned. A minimum-evolution phylogenetic tree was constructed based on the nucleotide sequences of exon 1 of Pib in 34 accessions and partial regions of exon-3 nucleotide sequences (from 7633 to 8484 of AB013448.1) of Pib in 49 accessions, respectively (Fig. 4). Exon 1 of Pib in wild-rice species (O. rufipogon, O. meyeriana, O. officinalis, O. longistaminata) was close to that in O. sativa (Fig. 4B). The DQ317978.1 group of the wild rice O. rufipogon shared >90% identity with the nucleotide sequences of the JN564624.1 group of Indica. Two major clades emerged in one part of exon 3 of Pib (Fig. 4C). One clade contained two wild-rice species (O. rufipogon and O. nivara) and O. sativa. The EF642422.1 group of the wild-rice species O. nivara shared >90% identity in nucleotide sequences with the EF642423.1 group of O. sativa. The EF642442.1 group of O. rufipogon shared >75% identity of nucleotide sequences with the EF642423.1 group of O. sativa. The other clade contained O. rufipogon and O. sativa. The EF642440.1 group of the wild-rice species O. rufipogon shared >75% identity of nucleotide sequences with the EF642433.1 group of O. sativa. The isolate of YN441 (with the H9 haplotype of AVR-Pib) was virulent to O. rufipogon, O. meyeriana, and O. officinalis (Fig. 3). These results suggested that different regions of the Pib gene may have suffered different selection pressures in the host rather than domestication.

We identified 12 new haplotypes, as well as Pot2 and Pot3 insertion in the AVR-Pib DNA sequences among rice-blast isolates from different rice-growing areas in Yunnan Province. The many virulent isolates to Pib-containing rice varieties implied that Pib was overcome in these rice-growing regions because of massive exploitation of Pib in China.

Pib alleles have been used widely and have shown strong resistance to disease in China [18]. Complete deletions have occurred in AVR-Pib sequences among field isolates of M. oryzae from various rice-growing regions of Guangdong, Hunan, and Liaoning Provinces [3]. Also, TE insertion has occurred in AVR-Pib in M. oryzae isolates from south and northeast China [3] and the Philippines [13]. Those data are consistent with our results. The L1 genotype of AVR-Pib identified in rice-blast isolates collected from rice fields implied that Pib has been effective in preventing rice blast. Li and colleagues showed that rice cultivars with Pib were resistant to 74.9% of isolates (282 isolates) from Yunnan Province [26]. The corresponding value was 2.1% in Guangdong Province (146 isolates were tested) [28], and the percentage resistance was <31% in Hunan Province [29]. These results show that Pib alleles had poor effects in these rice-production areas. Further inspection of variation in AVR-Pib DNA sequences in these isolates could reveal the molecular evolutionary patterns of AVR-Pib and predict the durability and effectiveness of Pib allele-mediated resistance under field conditions in rice-production regions.

AVR-Pia, AVR-Pii and AVR-Pita1 located on telomere regions tend to be unstable, and effective mutants in these genes have been identified [6, 30–31]. The retrotransposon (MINE) insertion in the ACE1 gene [8] and Pot3 insertion in AVR-Pita1 [32–33] and AVR-Pizt [7] have caused new virulent alleles. TE (Pot2 and Pot3) insertion, complete absence, segmental deletion, and a point mutation have been found in AVR-Pib alleles, all of which lead to gain of virulence [3]. Three expression patterns have been identified among different haplotypes of AVR-Pib [3]. Recently, insertion of a Pot3 transposon in AVR-Pib was shown to mediate the loss of function of AVR-Pib in all 248 isolates collected from the Philippines [13]. These findings showed that rice blasts can use transposons to suppress expression of AVR genes to defeat the rice-blast resistance gene. The AVR-Pib gene was identified in nearly half of rice-blast isolates (44.3%) in Yunnan Province (Table 1). This percentage was higher than that in rice-blast isolates in Jilin and Heilongjiang Provinces, but lower than those in Guangdong, Hainan and Liaoning Provinces [3]. Meanwhile, 28.6% of isolates contained a TE insertion in AVR-Pib. Among them, 21.4% isolates contained a Pot3-element insertion and 2.4% of isolates contained a reverse Pot3-element insertion, and 4.8% isolates contained a Pot2-element insertion in AVR-Pib of M. oryze from Yunnan Province (Table 4; Fig. 1). These insertions resulted in variation from avirulence to virulence to the corresponding R gene. Several nucleotide variations in AVR-Pib alleles were identified, which led to variations in amino acids and implied that AVR-Pib alleles suffer strong selection pressure in rice-production regions of Yunnan Province.

We observed no TE insertions in AVR-Pib of isolates from E. indica or E. coracana, and TE insertion in AVR-Pib was selected by the host, data that are consistent with the results of Zhang et al. [3]. Pot2 and Pot3 insertions were identified in the XI-rice growing areas, whereas only Pot3 insertion was identified in GJ-rice growing areas of Yunnan Province. TE insertion of AVR-Pib was noted in all rice-growing regions except northwestern Yunnan Province, and Pot3 insertion was distributed mainly in western Yunnan Province. These results showed that the virulent AVR-Pib alleles had been involved in most of the rice-growing regions of Yunnan Province. Hence, monitoring of these virulent alleles in field populations is important for employing Pib-containing rice varieties.

Various mutations were identified in CDS regions of AVR-Pib, and 12 AVR-Pib haplotypes were found based on 18 variant nucleotides among 90 isolates of L1 alleles collected from Yunnan Province (Table 2). Six new variant amino acids of the AVR-Pib loci variants were identified in the 90 M. oryze isolates in the present study, and resulted in identification of four novel haplotypes. A more holonomic network was constructed based on the new variations among different alleles of AVR-Pib. The putative secreted proteins of AVR-Pib in 126 isolates were identified (Table 3), and they were in accordance with the results of Zhang et al. [3]. Nine isolates had variations at the amino-acid position F54L; three isolates had variations at the amino-acid positions of E46V, Y53S, and F54V; one isolate had variations at the amino-acid positions of F47L, I49T, and R50G (Table 3). These isolates were virulent to the monogenic line IRBLb-B (with Pib), suggesting that these amino acids are crucial for avirulent function.

In the course of interactions and co-evolution between pathogens and plants, the R genes of plants can discern the cognate AVR genes of pathogens and inspire immunity. The genetic variation of the AVR genes of the pathogen is dependent upon the R genes of the host and changeable environmental conditions. The DI of AVR-Pib was higher in the GJ rice-production areas than that of XI rice-production areas (Table 4). Different variations were observed in AVR-Pib between XI rice- and GJ rice-production areas (Table 4). These findings imply that the adaptive mutations of AVR-Pib occurred in Yunnan Province under natural conditions.

Yunnan Province is abundant in genetic resources of rice. The wild species of O. officinalis, O. meyeriana and O. rufipogon also coexist in this Province [34]. More than 5000 rice accessions germplasms have been conserved in Yunnan Province. Among them, 12 out of 227 accessions carried the Pib resistance gene screened by resistance gene identification using different isolates [34]. Pib gene homologs have been identified in wild rice O. rufipogon from Yuanjiang County [20], and four genotypes (L0 to L3) of AVR-Pib have been detected in M. oryzae and O. rufipogon in Yuanjiang County. TE insertion of L2 and L3 genotypes of AVR-Pib was absent in the isolates from D. sanguinalis and M. nana Lour. These results suggest that adaptive variation of AVR-Pib is involved during interactions and co-evolution between AVR-Pib of M. oryzae and Pib of O. rufipogon. The Tajima’s D value of −1.61687 (Additional file 1: Table S1) indicates that AVR-Pib loci may suffer purifying selection by the corresponding R gene in Yunnan rice-production areas.

Massive variations and stepwise mutations in AVR-Pib of rice-blast isolates were observed in Yunnan Province (Table 2; Fig. 2), which suggests that there is abundant diversity of rice accessions and M. oryzae isolates in Yunnan Province. Pot2 insertion in AVR-Pib was found in western, southwestern, and northeastern Yunnan Province. Pot3 reversed-insertion was found in central and northeastern Yunnan Province. Also, Pot3 insertion occurred mainly in western Yunnan Province. Pot3 insertion of AVR-Pib was found in GJ rice- and XI rice-production areas, whereas Pot2- and Pot3-reverse insertion was found only in the XI rice-production area and GJ rice-production area, respectively (Table 4). The virulent haplotype of H11 was detected in the XI rice-production area and GJ rice-production area, and H08 was detected in the XI rice-production area. These data showed high variation of AVR-Pib in different rice-growing regions, which may be due to rice variety and the environment.

The stepwise mutations that result in loss of avirulence function have been identified in AVRL567 [35] and AVR-Pik [36–38]. Based on the Pib homologs identified by Yang et al. [20], and our result for AVR-Pib in the present study, the potential interactions and co-evolution of AVR-Pib alleles in M. oryzae and Pib alleles of rice were constructed (Additional file 9: Fig. S6). The AVR-Pib homolog L1 (H01) originated from an ancestral M. oryzae gene. The Pib allele (87-bp deletion in exon 1 of Pib) in O. rufipogon could not recognize the L1 alleles of AVR-Pib. Thus, the other Pib allele (gained 87 bp in exon 1 of Pib) in cultivated rice evolved to recognize the L1 alleles (H01) of AVR-Pib, whereas the altered alleles L2 and L3 evolved to virulence from avirulent origins by TE insertion, base substitution (H08) and segment insertion (H11) to avoid recognition by Pib (Table 2; Additional file 9: Fig. S6). These actions indicated stepwise evolution of AVR-Pib as well as Pib interaction and co-evolution. Intriguingly, the AVR-Pib alleles H08 and H11 were derived from H01, and could escape recognition by Pib (Table 2; Additional file 9: Fig. S6), but several extinct or missing haplotypes were not identified in the sample (Fig. 2). These findings imply that: (i) the AVR-Pib loci of M. oryzae evolved gradually during the interaction and coevolution between the Pib loci of M. oryzae in field conditions; (ii) the genome organization of the AVR-Pib locus is much more intricate than anticipated.

We detected twelve novel haplotypes in the field population by using 90 isolates, transposable element (TE) insertion in 36 of 126 isolates, constructed a complex network of AVR-Pib alleles, and assessed the efficacy of Pib alleles in rice production areas of Yunnan, analyzed the adaption of TE insertion of AVR-Pib in the isolates from different host. Our findings support the hypothesis that functional AVR-Pib possesses varied sequence structures and can escape surveillance by hosts via multiple variation manners. Haplotype H08 and H11 can overcome all detected Pib alleles to date, and Pot insertion can changed the avirulent function of AVR-Pib. Despite the H08, H11 haplotypes and TEs insertions have low frequencies, monitoring of these alleles in field populations is critical because of their high risk for Pib-holding rice varieties. The TE insertion was not detected in AVR-Pib allele in the isolates from E. indica, E. coracana, and Triticum aestivum Linn, while three genotypes of AVR-Pib were detected in isolates from O. rufipogon, the selected and adapted variation of TE insertion in AVR-Pib is occurrence in the long-term co-evolution between M. oryzae and hosts (rice and other Gramineae species).

AVR

Avirulence gene

Diversity index

Geng/Japonica

The rate of nonsynonymous substitution

The rate of synonymous substitution

LTH

Lijiangxintuanheigu

Resistance

Moderate resistant

Susceptible

Transposable element

Xian/Indica

Blast isolates, rice accessions, culture, and pathogenicity identification

The seedlings of the rice monogenic line IRBLb-B (which contains Pib) and a susceptible cultivar Lijiangxintuanheigu (LTH; does not contain Pib) were used for pathogenicity assays (the seeds were acquired originally from Cailin Lei). The total of 366 isolates of M. oryzae in the present study were same with the published paper [38]. The storing and culturing methods of the isolates were described in the published paper [38]. Disease reactions were referred as the method of Jia et al. [39]. In a few words, when rice seedlings were at the 3- to 4-leaf stage, they were inoculated with a spore suspension (1–5×10⁵spores/mL containing 0.05% Tween 20). After innoculation, rice seedlings were put into a plastic bag and sealed securely for keeping high relative humidity of 90 -100% at 25°C for 24 h in the dark. Then, the plants were shifted into a greenhouse for 6 days to develop disease lesion extension fully.

Disease reactions were monitored externally on the second youngest leaf based on the number and degree of lesions using a 0-to-5 disease scale (Additional file 10: Fig. S7). The disease scale method was described in the published paper [38] Five seedlings at each time and twice repeats were arranged in the experiment. And the average value of disease scales was used to discrinate resistance versus susceptibility. The disease reaction of wild rice O. rufipogon, O. meyeriana, and O. officinalis was determined using a detached-leaf method, as described by Jia et al. [39]. One virulent blast isolate was used for inoculation. Disease reactions were evaluated 5–7 days after inoculation.

DNA extraction, PCR amplification, and DNA sequencing

Culturing method of vegetative mycelia of M. oryzae isolates were described in the published paper [38]. The genomic DNA of each isolate was extracted from vegetative mycelia by CTAB method [40]. The primers AvrPibF1 (5′-GGACAAGGGAGGCAAATCTAAC-3′) and AvrPibR1 (5′-ATGCCGACAATGCGAGGTAT-3′) were used to amplify the AVR-Pib allele and for sequencing according to the method of Zhang et al. [3]. Each PCR reaction was amplified in a total reaction volume of 50 µL containing the following components: 25 µL of 2× Taq PCR MasterMix (Tiangen Biotech, Beijing, China), 1 µL (10 µM) of each primer, 2 µL of template DNA, and 21 µL of ddH₂O (provided in the Tiangen kit). PCRs procedure were done in a C1000 Touch™ thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) as the following steps: initial denaturation at 94°C for 3 min, followed by 29 cycles at 94°C for 45 s, 55°C for 45 s, and 72°C for 2.5 min, and a final extension at 72°C for 5 min. Each reaction was repeated twice. The size of the PCR products was valued by a DNA marker (DL2000, Tiangen Biotech). Amplicons were sequenced twice by Life Technologies Biotechnology (Shanghai, China).

Data analyses

DNA sequences of AVR-Pib were assembled and aligned using DNASTAR v7.1.0 (www.dnastar.com/). DnaSP v5.10.01 [41] was used for calculation of polymorphic sites (π), the number of DNA haplotypes, and the sliding window. TCS1.21 (http://darwin.uvigo.es/) [42] was used for analyses of the haplotype network of AVR-Pib. The DI (haplotype diversity index) was counted in M. oryzae populations following the method of Fontaine et al. [43]:

DI = (1 − ∑ⁿ_{i = 1}p_i²)

where p_i is the frequency of haplotype i in a population. Tajima’s neutrality test was done using MEGA X (www.megasoftware.net) [44]. The Selection Server program (http://selecton.tau.ac.il) was used for analyses of purifying selection. The purifying selected sites of AVR-Pib was identified by used three models: M8 (positive selection enabled, beta + w ≥ 1), M7 (beta, null model) and M8a (beta + w = 1, null model). Then, the sliding window of purifying selected sites of AVR-Pib was draw under the M8, M7, and M8a models by Excel™ (Microsoft, Redmond, WA, USA). MEGA X [44] was used for construction of phylogenetic trees by the minimum-evolution method [45]. The SWISS-MODEL (http://swissmodel.expasy.org) with ProMod v3.7.0 was used to built the protein homology model. The significant difference of distribution of AVR-Pib genes and avirulence isolates of M. oryzae in each region was analyzed by Excel software with CHITEST.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the Key Projects of Yunnan Province (2017FA013), the National Natural Science Foundation of China (31860481), the Key Research and Development Program of Yunnan Province (2019IB007 and 202102AE090003), the program of the Innovative Research Team of Yunnan Province (202005AE160003), the Key Laboratory of Green Prevention and Control of Agricultural Transboundary Pests of Yunnan Province (202105AG070001). The funding organizations had no role in designing, data collection, analysis, and interpretation of data and in writing the manuscript.

Authors’ contributions

JBL was the leading investigators of this research programme. JBL planned and designed the research; JBL, QW and LL performed the majority of experiments with the help of CYL; LL, ZFS and CYL contributed reagents, materials, and analysis tools; JBL and QW analyzed the data; JBL and QW wrote the paper with suggestions from LL, CYL and ZFS. All authors commented on the article before submission. The author(s) read and approved the final manuscript.

Acknowledgments

The authors thank Professor Zaiquan Cheng (Institute of Biotechnology and Genetic Resources, Yunnan Academy of Agricultural Sciences) and Professor Yiqing Guo (Yunnan Agricultural University) for useful discussions.

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10 Mar, 2022
First submitted to journal
19 Dec, 2021

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Insertion of transposable elements in AVR-Pib of Magnaporthe oryzae

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