Potyviral resistance derived from cultivars of Phaseolus vulgaris carrying bc-3 is associated with the homozygotic presence of a mutated eIF4E allele

Abstract

Eukaryotic translation initiation factors (eIFs) play a central role in potyviral infection. Accordingly, mutations in the gene encoding eIF4E have been identified as a source of recessive resistance in several plant species. In common bean, Phaseolus vulgaris, four recessive genes, bc-1, bc-2, bc-3 and bc-u, have been proposed to control resistance to the potyviruses Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus. In order to identify molecular entities for these genes, we cloned and sequenced P. vulgaris homologues of genes encoding the eIF proteins eIF4E, eIF(iso)4E and nCBP. Bean genotypes reported to carry bc-3 resistance were found specifically to carry non-silent mutations at codons 53, 65, 76 and 111 in eIF4E. This set of mutations closely resembled a pattern of eIF4E mutations determining potyvirus resistance in other plant species. The segregation of BCMV resistance and eIF4E genotype was subsequently analysed in an F(2) population derived from the P. vulgaris all-susceptible genotype and a genotype carrying bc-3. F(2) plants homozygous for the eIF4E mutant allele were found to display at least the same level of resistance to BCMV as the parental resistant genotype. At 6 weeks after inoculation, all F(2) plants found to be BCMV negative by enzyme-linked immunosorbent assay were found to be homozygous for the mutant eIF4E allele. In F(3) plants homozygous for the mutated allele, virus resistance was subsequently found to be stably maintained. In conclusion, allelic eIF4E appears to be associated with a major component of potyvirus resistance present in bc-3 genotypes of bean.

Figure 1

Comparison of amino acid polymorphisms in eukaryotic translation initiation factor 4E (eIF4E) found between susceptible and resistant/tolerant genotypes of Capsicum annuum (pvr1+, AY485127; pvr1, AY485129; pvr1¹, AY485130; pvr1², AY485131), Lactuca sativa (mo1+, AAP86602; mo1¹; mol²) and Pisum sativum (sbm1+, AAR04332; sbm1, AAT44121; sbm1¹, AAT44122) with eIF4E encoded by Phaseolus vulgaris alleles PveIF4E¹ (EF571267) and PveIF4E² (EF571275). PveIF4E² found in the genotypes carrying the bc‐3 gene. The amino acids conserved in susceptible alleles of all four species are shaded in grey. Codons mutated in PveIF4E² are indicated by numbers and similarly located mutations in eIF4E genes linked to potyvirus resistance in the other species are framed by rectangles. The symbol Δ indicates the amino acids deleted.

Figure 2

Segregation of Bean common mosaic virus (BCMV) resistance and a cleaved amplified polymorphic sequence (CAPS) marker differentiating eIF4E alleles PveIF4E² (4E²) and PveIF4E¹ (4E¹). A Phaseolus vulgaris F₂ population was generated from a cross between parents (P) USCR8 (4E²/4E², lane 2) and Dubbele Witte (DW) (4E¹/4E¹, lane 3). Successful crossing was verified on F₁ individuals (lane 4) and results from 17 F₂ individuals are shown (lanes 5–21).The response to BCMV was assayed by enzyme‐linked immunosorbent assay (ELISA). Plants with an ELISA A405 (absorbance at 405 nm) reading of more than 2.5 times that of the mock‐inoculated control were rated as susceptible (s); plants with an ELISA A405 reading of less than 2.5 times that of the mock‐inoculated control were rated as resistant (r). The genotype was determined by restriction enzyme RsaI digestion of a 541‐bp polymerase chain reaction (PCR) product amplified with primers ENM‐FWe and ENM‐RVe from genomic DNA. DNA fragments were analysed by agarose gel electrophoresis with a 100‐bp ladder as marker (lane 1).

Figure 3

Average Bean common mosaic virus (BCMV) antigen levels in parental lines and F₂ plants grouped by eIF4E genotype. In two experiments, A and B, plants were inoculated with BCMV strain NL1 and analysed by BCMV enzyme‐linked immunosorbent assay (ELISA) of the top leaves at 6 weeks post‐inoculation (wpi). Dark columns show the average BCMV antigen levels and the bars on top indicate the standard deviation within each group. The ELISA control (CON, white column) is the average of two non‐inoculated plants of USCR8. The PveIF4E genotype was determined by application of the RsaI cleaved amplified polymorphic sequence (CAPS) marker, and plants were grouped into homozygous PveIF4E¹ (4E¹/4E¹), homozygous PveIF4E² (4E²/4E²) and heterozygous (4E¹/4E²) plants. The number of plants in each group is indicated.

Figure 4

Average Bean common mosaic virus (BCMV) absorbance ratios in parental genotypes, F₂ plants homozygous for PveIF4E² (4E²/4E²) and F₃ plants derived from 4E²/4E² F₂ plants. (A) 4E²/4E² F₂ individuals (dark columns) and parental USCR8 plants (USCR8, grey columns) from experiments 1 and 2 are divided into enzyme‐linked immunosorbent assay (ELISA)‐positive (+) and ELISA‐negative (–) groups and compared with parental cultivar Dubbele Witte (DW) (all‐susceptible, grey column). (B) F₃ plants (dark columns) derived from ELISA‐positive (+) and ELISA‐negative (‐) 4E²/4E² F₂ (F₂+ and F₂–, respectively) plants were inoculated with BCMV strain NL1 and analysed by BCMV ELISA of the top leaves at 6 weeks post‐inoculation (wpi). F₃ plants within each group were subdivided into ELISA positive (+) and ELISA negative (–) on the basis of the absorbance ratio. Columns show the absorbance ratios and the bars on top indicate the standard deviation within each group. The ELISA control (CON, white column) is the average of two non‐inoculated plants of USCR8. The number of plants in each group is indicated.