The origin and distribution of 'Kokubu'-type splice-site mutations of the MLO genes in tobacco varieties.

The Japanese domestic tobacco (Nicotiana tabacum L.) cultivar 'Kokubu' shows high powdery mildew resistance that is controlled by splice-site mutations of two MILDEW LOCUS O genes, NtMLO1 and NtMLO2. We investigated the existence of the same NtMLO1/2 splice mutations in the genomes of various tobacco varieties cultivated in Japan and other countries. In total, 14 Japanese domestic cultivars, which were mainly distributed in Kagoshima, had splice-site mutations in both NtMLO1 and NtMLO2. In addition, tobacco cultivars containing only the NtMLO1 splice-site mutation were found in various tobacco production areas in Japan, but no cultivars with only the NtMLO2 splice-site mutation were detected. Moreover, the NtMLO1 splice-site mutation was detected in native Asian, Oriental and cigar tobacco varieties. Consequently, we speculate that these powdery mildew-resistant tobacco cultivars were generated relative recently in the Kagoshima area when a spontaneous mutation occurred at the NtMLO2 splice site in a cultivar already containing the NtMLO1 splice-site mutation and that the NtMLO1 splice-site mutation occurred during the early period of tobacco seed dissemination from the Americas to Asia and Japan.

Loss-of-function of Nicotiana tabacum L. eukaryotic translation initiation factors eIF4E1-S and eIF(iso)4E-T synergistically confers high-level resistance to both Potato virus Y (PVY) and resistance-breaking PVY.

The plant eukaryotic translation-initiation factors eIF4E and eIF(iso)4E play key roles in infection by plant RNA viruses, especially potyviruses. Mutations in the genes that encode these factors reduce susceptibility to the viruses. In the amphidiploid plant tobacco (Nicotiana tabacum L.), eIF4E1-S deletion mutants resist Potato virus Y (PVY), but resistance-breaking strains (RB-PVY) have appeared. In an earlier study, we demonstrated that the loss-of-function of eIF(iso)4E-T reduces susceptibility to RB-PVY. Here, we show that simultaneous inhibition of eIF4E1-S and eIF(iso)4E-T synergistically confers enhanced resistance to both PVY and RB-PVY without host growth or development defects. PVY symptoms and accumulation in a tobacco line lacking eIF4E1-S were detected at 14 days post-inoculation (dpi) and RB-PVY symptoms in lines without functional eIF(iso)4E-T were observed at 24 dpi. RB-PVY emerged in a PVY-infected tobacco line lacking eIF4E1-S. In contrast, lines without functional eIF4E1-S and eIF(iso)4E-T were nearly immune to PVY and RB-PVY, and little accumulation of either virus was detected even at 56 dpi. Thus, the lines will be promising for PVY-resistance breeding. This study provides a novel strategy to develop tobacco highly resistant to PVY and RB-PVY, and insights into the mechanisms responsible for high-level resistance.

Highly Efficient and Comprehensive Identification of Ethyl Methanesulfonate-Induced Mutations in Nicotiana tabacum L. by Whole-Genome and Whole-Exome Sequencing.

Tobacco (Nicotiana tabacum L.) is a complex allotetraploid species with a large 4.5-Gb genome that carries duplicated gene copies. In this study, we describe the development of a whole-exome sequencing (WES) procedure in tobacco and its application to characterize a test population of ethyl methanesulfonate (EMS)-induced mutations. A probe set covering 50.3-Mb protein coding regions was designed from a reference tobacco genome. The EMS-induced mutations in 19 individual M2 lines were analyzed using our mutation analysis pipeline optimized to minimize false positives/negatives. In the target regions, the on-target rate of WES was approximately 75%, and 61,146 mutations were detected in the 19 M2 lines. Most of the mutations (98.8%) were single nucleotide variants, and 95.6% of them were C/G to T/A transitions. The number of mutations detected in the target coding sequences by WES was 93.5% of the mutations detected by whole-genome sequencing (WGS). The amount of sequencing data necessary for efficient mutation detection was significantly lower in WES (11.2 Gb), which is only 6.2% of the required amount in WGS (180 Gb). Thus, WES was almost comparable to WGS in performance but is more cost effective. Therefore, the developed target exome sequencing, which could become a fundamental tool in high-throughput mutation identification, renders the genome-wide analysis of tobacco highly efficient.

DNA marker development by the allele-specific detection of powdery mildew resistance loci derived from Japanese domestic tobacco cultivar 'Kokubu'.

Japanese domestic tobacco (Nicotiana tabacum L.) cultivar 'Kokubu' shows high powdery mildew resistance controlled by recessive alleles at two loci, and these alleles have been widely used as a resource for powdery mildew resistance in tobacco breeding. However, the introduction of this trait by conventional breeding takes much work because of the requirement for test crosses with the parental strains and inoculation tests using active fungi to confirm the introduction of two recessive alleles during back-crossing. Recently, we found that powdery mildew resistance in 'Kokubu' is caused by splice site mutations of two MILDEW LOCUS O genes, NtMLO1 and NtMLO2. Here, we report DNA markers that detect mutations of the NtMLO1/2 genes based on the cleaved amplified polymorphic sequence (CAPS) or allele-specific polymerase chain reaction (AS-PCR) methods. These markers can be used as co-dominant markers that detect heterozygotes of the NtMLO genes at the seedling stage in back-crossed progenies, and will contribute to the simplification of breeding.

Reduced susceptibility to a tobacco bushy top virus Malawi isolate by loss of function in host eIF(iso)4E genes.

Tobacco bushy top disease (TBTD) is a viral disease of tobacco (Nicotiana tabacum L.) caused by mixed infection of Tobacco bushy top virus or Ethiopian tobacco bushy top virus and a helper virus. Despite its damage to tobacco, practical genetic resources for disease resistance have not been found. Here, we report that a mutation of tobacco eIF(iso)4E genes (eIF(iso)4E-S and eIF(iso)4E-T), which encode eukaryotic translation initiation factors, confers resistance (reduced susceptibility) to TBTD caused by a virus from Malawi (designated as tobacco bushy top virus Malawi isolate, TBTV-MW). RNAi lines in which eIF(iso)4E genes were silenced showed reduced susceptibility to TBTV-MW. We also tested chemically-induced single (eIF(iso)4E-S or eIF(iso)4E-T) and double (eIF(iso)4E-S and eIF(iso)4E-T) nonsense mutants for resistance to TBTV-MW. Suppression of eIF(iso)4E-S showed reduced susceptibility, and the resistance of the double mutant tended to be even stronger. eIF(iso)4E mutants also showed reduced susceptibility to TBTV-MW transmitted by aphids. To the best of our knowledge, the eIF(iso)4E-S mutant is the first genetic resource for TBTD resistance breeding in tobacco.

Mutation of a Nicotiana tabacum L. eukaryotic translation-initiation factor gene reduces susceptibility to a resistance-breaking strain of Potato virus Y.

Eukaryotic translation-initiation factors eIF4E and eIF(iso)4E in plants play key roles in infection by potyviruses and other plant RNA viruses. Mutations in the genes encoding these factors reduce susceptibility to the viruses, and are the basis of several recessive virus resistance genes widely used in plant breeding. Because virus variants occasionally break such resistance, the molecular basis for this process must be elucidated. Although deletion mutants of eIF4E1-S of tobacco (Nicotiana tabacum L.) resist Potato virus Y (PVY; the type member of the genus Potyvirus), resistance-breaking strains of PVY threaten tobacco production worldwide. Here, we used RNA interference technology to knock down tobacco eIF4E2-S and eIF4E2-T genes or eIF(iso)4E-S and eIF(iso)4E-T genes. Transgenic plants with reduced transcript levels of both eIF(iso)4E-S and eIF(iso)4E-T showed reduced susceptibility to a resistance-breaking PVY strain with a K105E mutation in the viral genome-associated protein (VPg). By screening a population of chemically induced mutants of eIF(iso)4E-S and eIF(iso)4E-T, we showed that plants with a nonsense mutation in eIF(iso)4E-T, but not eIF(iso)4E-S, showed reduced susceptibility to the resistance-breaking PVY strain. In a yeast two-hybrid assay, VPg of the resistance-breaking strain, but not wild-type PVY, physically interacted with the eIF(iso)4E-T protein. Thus, eIF4E1-S is required for infection by PVY, but eIF(iso)4E-T is required for infection by the resistance-breaking strain. Our study provides the first evidence for the involvement of a host eukaryotic translation-initiation factor in the infection cycle of a resistance-breaking virus strain. The eIF(iso)4E-T mutants will be useful in tobacco breeding to introduce resistance against resistance-breaking PVY strains.

The self-compatibility mechanism in Brassica napus L. is applicable to F1 hybrid breeding.

Brassica napus, an allopolyploid species having the A genome of B. rapa and the C genome of B. oleracea, is self-compatible, although both B. rapa and B. oleracea are self-incompatible. We have previously reported that SP11/SCR alleles are not expressed in anthers, while SRK alleles are functional in the stigma in B. napus cv. 'Westar', which has BnS-1 similar to B. rapa S-47 and BnS-6 similar to B. oleracea S-15. This genotype is the most frequent S genotype in B. napus, and we hypothesized that the loss of the function of SP11 is the primary cause of the self-compatibility of 'Westar'. To verify this hypothesis, we transformed 'Westar' plants with the SP11 allele of B. rapa S-47. All the transgenic plants and their progeny were completely self-incompatible, demonstrating self-compatibility to be due to the S haplotype having the non-functional SP11 allele in the A genome, which suppresses a functional recessive SP11 allele in the C genome. An artificially synthesized B. napus line having two recessive SP11 alleles was developed by interspecific hybridization between B. rapa and B. oleracea. This line was self-incompatible, but F(1) hybrids between this line and 'Westar' were self-compatible. These results suggest that the self-compatibility mechanism of 'Westar' is applicable to F(1) seed production in B. napus.