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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.


Introduction
Tobacco bushy top disease (TBTD) is a viral disease that damages tobacco (Nicotiana tabacum L.) in Africa and China (Abraham et al. 2014, Gates 1962, Mo et al. 2002. Infected tobacco shows symptoms such as growth delay, mottling (often with spotted necrosis/chlorosis), and abnormal sucker growth, which reduce quality and yield. Tobacco bushy top virus (TBTV), reported in China, and Ethiopian tobacco bushy top virus (ETBTV), reported in Ethiopia, both cause TBTD (Abraham et al. 2014, Mo et al. 2003. Based on the features of genomic RNA structure and/or viral morphology observed by an electron microscope, both viruses are categorized in the genus Umbravirus (Abraham et al. 2014, Mo et al. 2003, Taliansky and Robinson 2003. Umbraviruses, generally, have four major open reading frames (ORFs); ORF1 and ORF2 code for RNA dependent RNA polymerase, ORF3 is responsible for long-distance movement, and ORF4 is responsible for cell to cell movement (Gibbs et al. 1996, Mo et al. 2003, Ryabov et al. 1999, Taliansky et al. 1996. As umbraviruses lack ORFs for the coat protein, they are unable to form virions by themselves (Falk et al. 1979, Murant et al. 1969. Instead, a coat protein for the umbraviruses is provided by a helper virus of the family Luteoviridae in the natural environment, where umbraviruses are transmitted via aphids (Demler et al. 1994, Murant et al. 1985. For example, Tobacco vein distorting virus and Potato leafroll virus are helper viruses for TBTV and ETBTV, respectively (Abraham et al. 2014, Gates 1962, Mo et al. 2010.
The management of aphids is one practical way to control TBTD (Abraham 2019). Pesticides can reduce aphids directly, and, by adjusting the transplantation period, tobacco can be cultivated avoiding the timing of the virus infection by aphids. However, these cultivated ways are much laborious and costly. The development of resistant cultivars is another way to reduce damages by the virus. Although the genetic resources that might show resistance to TBTD have been reported in wild Nicotiana relatives (Bruin 1990, Gates 1962, resistant cultivars have never been developed in N. tabacum to date. Studies on virus-host interactions provide clues to virus resistance. Viral proteins or genome of certain viruses interact with eukaryotic translation initiation factors (eIFs) in host-plants for their replication and for cell-to-cell movement (Thivierge et al. 2005). Many potyviruses interact with eIF4E, a host cap-binding protein, or its isoform eIF(iso)4E, through their viral genome-associated protein (VPg) Caranta 2006, Wittmann et al. 1997). The 5ʹ leader of Tobacco etch virus (genus Potyvirus) genomic RNA and the VPg of Rice yellow mottle virus (genus Sobemovirus) interact directly with eIF4G, a scaffold protein for other eIFs, and its isoform eIF(iso)4G, respectively (Gallie 2001, Hébrard et al. 2010. Since eIFs are factors involved in the susceptibility of hosts, natural variation or artificially induced mutations in eIF genes enable to reduce the degree of susceptibility (Albar et al. 2006, Bastet et al. 2017, Diaz-Pendon et al. 2004, Nieto et al. 2006, Piron et al. 2010, Yoshii et al. 2004). In the case of tobacco, resistance to potato virus Y (PVY), a member of genus Potyvirus, which causes one of the most serious diseases of tobacco, was conferred by the deletion of eIF4E (Julio et al. 2015), and the deletion is widely used in tobacco breeding. Although Resistance-Breaking PVY (RB-PVY) strains can break this resistance (e.g., Masuta et al. 1999), our group recently reported that the VPgs of RB-PVY strains interacted with eIF(iso)4E in their infection cycle and that a nonsense mutation of eIF(iso)4E conferred RB-PVY resistance (Takakura et al. 2018).
N. tabacum is an allopolyploid (amphidiploid) species that combines the S and T genomes derived from ancestral Nicotiana sylvestris and Nicotiana tomentosiformis. Genomic analysis revealed that N. tabacum has both S genome derived type (S-type) and T genome derived type (T-type) of eIF4E1, eIF4E2, and eIF(iso)4E (Sierro et al. 2014). In the present study, we investigated the involvement of tobacco eIF4E and eIF(iso)4E in the susceptibility to a virus causing TBTD in Malawi (tobacco bushy top virus Malawi isolate, TBTV-MW) to find genetic resources for TBTD resistance.

Virus, sequencing of viral genome, and assembly
TBTV-MW derived from infected leaf collected in Malawi has been maintained by recurrent sap inoculation into tobacco (N. tabacum) cv. Tsukuba 1 (we used "tobacco bushy top virus Malawi isolate" or "TBTV-MW" as the virus isolate name in the present study, but not as the species name, because classification of the virus isolate was not clear). Total RNA was extracted by the use of an RNeasy kit (QIAGEN) from infected leaves of plants showing bushy top symptoms, and tobacco DNA was degraded by a Turbo DNA-free Kit (Thermo Fisher Scientific). Following library preparation by a TruSeq Stranded Total RNA with Ribo-Zero Plant Kit (Illumina), sequencing was performed with a NextSeq 500/550 Mid Output Kit v2 (2 × 150 bases, Illumina). Next-generation sequencing data was processed in CLC Genomics Workbench v. 11.0 software. Trimmed reads were mapped to the tobacco cv. K326 reference genome (GenBank accession PRJNA208210), and contigs were obtained by de novo assembly of unmapped reads. Nucleotide sequence data of TBTV-MW genome has been deposited in DDBJ/EMBL/GenBank databases with the LC494673 accession code.

Preparation of RNAi-tobacco transformants
We generated eIF4E1-RNAi lines by the same procedure as Takakura et al. (2018): eIF4E1-S (GenBank accession KF155696) was amplified from cDNA of 'Tsukuba 1' leaf with the primers in Supplemental Table 1. The amplicon was purified by using a MinElute PCR Purification Kit (QIAGEN). After confirmation of sequences as eIF4E1-S, the amplified fragment was cloned into pENTR/D-TOPO vector (Life Technologies). The amplicon was inserted into the pSP231 binary vector, which has a green fluorescent protein expression cassette in pHELLSGATE12 Waterhouse 2003, Wesley et al. 2001), by the use of Gateway LR Clonase II Enzyme Mix (Life Technologies). An eIF4E1-RNAi construct in which the trigger DNA fragments were cloned as a pair of inverted repeats driven by the Ca35S promoter was obtained by Gateway technology. Purified binary vector was transferred into Agrobacterium tumefaciens LBA4404 by electroporation, and the RNAi cassette was introduced into tobacco cv. SR1 Petit Havana by standard A. tumefaciens-mediated transformation. In T 1 progeny obtained by self-pollination of the primary transformants (T 0 ), the presence or absence of the cassette was checked by fluorescence in a ChemiDocMP Imaging System (Bio-Rad Laboratories). In each T 2 line, the presence or absence of the cassette was checked again by PCR of neomycin phosphotransferase II (Supplemental Table 1).

Preparation of tobacco mutants
eIF(iso)4E-S and eIF(iso)4E-T nonsense mutants (ssTT and SStt, with a point mutation in the first exon of the gene; Supplemental Fig. 1), the double mutant (sstt), and the wild-type segregant (SSTT) were selected by Takakura et al. (2018) from an ethyl methanesulfonate induced tobacco population. These plants had 'Tsukuba 1' genetic background. To prepare 'K326' background lines, the double mutant (sstt) was crossed with 'K326', followed by two more backcrosses, to generate BC 2 F 1 . After genotyping of BC 2 F 2 individuals, homozygotes for both eIF(iso)4E genes were self-pollinated, and the K326-ssTT, K326-SStt, K326sstt, and K326-SSTT lines were fixed at the BC 2 F 3 generation. Genotyping was performed by using derived Cleaved Amplified Polymorphic Sequence (dCAPS) maker as describe later.

RT-qPCR
Total RNA was extracted from leaf samples with an RNeasy kit (QIAGEN) and cDNA was synthesized with the use of a PrimeScript RT reagent Kit (Takara). RT-qPCR was performed using TaqMan Fast Advanced Master Mix (Life Technologies) and the primers and probes in Supplemental Table 1 to calculate the expression values of eIF genes and the accumulation of TBTV-MW. Elongation factor 1-α was used as an internal standard. Analysis was done in StepOne Real-Time PCR System v. 2.2 software (Life Technologies).

Sap inoculation test
TBTV-MW-infected 'Tsukuba 1' leaves were ground in two volumes of 0.01 N phosphate buffer (pH 7.0). The homogenate was inoculated with carborundum into half of the fifth or sixth leaf from the bottom in each tobacco seedling at about 30 days after sowing. Inoculated plants were grown in a greenhouse at 22 ± 2°C. The presence or absence of mottling symptoms in each plant was recorded to evaluate TBTV-MW resistance. For 'K326' background lines, another inoculated plant set different from the resistance evaluation test set was prepared. All the upper leaves other than the apical meristem were sampled at 13 days post-inoculation (DPI) to quantify the relative virus amount in the inoculated plants.

Aphid transmission test
Leaves were sampled from 'Tsukuba 1' infected with TBTV-MW and Tobacco vein distorting virus as a helper virus. Virus-free aphids (Myzus persicae) were fed on infected leaves for 1 day before transfer. Ten aphids per plant were placed on seedlings at 30 days after sowing, which was set as 0 DPI. Plants with aphids were grown in a growth chamber at 25°C, and aphids were removed at 7 DPI by a soil application of imidacloprid. Virus resistance was evaluated at 14, 19, and 25 DPI by observing the presence or absence of mottling symptoms. The topmost fully developed leaf of each plant was sampled at 25 DPI to quantify the virus amount.

dCAPS markers to detect eIF(iso)4E mutation
eIF(iso)4E-S and eIF(iso)4E-T were amplified by multiplex PCR with a mix of specific primers (Supplemental Table 1) and a Multiplex PCR kit (QIAGEN), which includes a DNA polymerase that does not have 3ʹ to 5ʹ exonuclease activity. Restriction enzymes NlaIII for eIF(iso)4E-S and MboI (both from New England Biolabs) for eIF(iso)4E-T were added to the PCR amplicons to digest each wild-type allele. Digested fragments were separated by electrophoresis in a QIAxcel System (QIAGEN), and genotypes were decided by fragment size.

Characterization of TBTV-MW genome and sequence comparison
Genomic RNA of TBTV-MW was sequenced by using next-generation sequencer. Out of 1,560 contigs generated by de novo assembly, one contig (#13) of 4,186 nt had 87.7% identity with genome sequences of ETBTV (GenBank accession KJ918748.1), 51.6% with that of TBTV (AF402620.2), and 59.7% with that of Groundnut rosette virus (Z69910.1) (Supplemental Table 2). No other contig showed distinct identity to known viruses. Amino acid sequences deduced from four predicted ORFs in the contig genome sequence also showed the highest identity (79.1%-94.6%) to those of ETBTV (Supplemental Table 2). Molecular phylogenetic analysis showed that the genome sequence of #13 contig was placed closest to that of ETBTV in the same cluster (Fig. 1). We concluded that the genome sequence of #13 contig corresponded to the TBTV-MW genome, and used it in the following experiments.

Reduced susceptibility to TBTV-MW by RNAi suppression of eIF(iso)4E
To find genes responsible for TBTV-MW resistance, we focused on six known eIF genes that encode eIF4E or eIF(iso)4E in tobacco, i.e., eIF4E1-S (GenBank accession KF155696), AY699609), and eIF(iso)4E-T (EB683576), and produced RNAi suppression lines for each gene. For eIF4E1-RNAi suppression lines, the RNAi cassette to suppress both Sand T-type of eIF4E1 genes at the same time was constructed. eIF4E2-and eIF(iso)4E-RNAi lines were previously prepared by Takakura et al. (2018). Expression levels of both eIF4E1s in leaves were evaluated by RT-qPCR in T 2 generations of each eIF4E1-RNAi transgenic line (Supplemental Fig. 2). The expression of both genes in each RNAi line was significantly reduced compared with that in non-transformants. The target genes were significantly suppressed in eIF4E2-RNAi and eIF(iso)4E-RNAi lines as well (Supplemental Fig. 2). Carrot mottle virus (CMoV) were clustered. The phylogenetic tree was drawn by the neighbor-joining method using the genome sequence of Sclerotinia sclerotiorum umbra-like virus 1 (SsULV1) as an outgroup in MEGA X software (Kumar et al. 2018). GenBank accession numbers are shown in parentheses.

Reduced susceptibility to TBTV-MW by nonsense mutation of eIF(iso)4E genes in the sap inoculation test
As eIF(iso)4E-RNAi(+) showed reduced susceptibility, we performed a sap-inoculation test for eIF(iso)4E mutants ('Tsukuba 1' background lines) to verify whether either or both of eIF(iso)4E-S and eIF(iso)4E-T are involved. In advance of the sap inoculation test, we measured expression levels of eIF(iso)4E-S and eIF(iso)4E-T in each singlenonsense mutant (ssTT and SStt), the double mutant (sstt), and the wild-type segregant (SSTT) by RT-qPCR. Transcription of the mutant gene was greatly reduced in each mutant, probably caused by nonsense-mediated mRNA decay (Nyikó et al. 2013, Shaul 2015 (Fig. 3A). The number of plants with symptoms was the most in SSTT and SStt getnotypes, followed by ssTT genotype. In sstt double mutant, the number was remarkably decreased (Fig. 3B,  3C).
To test the effect of the mutations in a different genetic background (e.g., Palloix et al. 2009, Quenouille et al. 2013), we prepared 'K326' background lines. The dCAPS markers were developed for detection of the wild-type and mutant alleles of both eIF(iso)4E-S and -T (Fig. 4A). After marker selection (Fig. 4B shows an example), RT-qPCR was carried out for the selected mutants. Reduced expres-sion of the mutant gene was confirmed in each 'K326' background line (Fig. 5A). Additionally, as in the 'Tsukuba 1' background lines, lines K326-sstt and K326-ssTT   Vol. 70 No. 3 Udagawa, Koga, Shinjo, Kitashiba and Takakura showed reduced susceptibility, while K326-SStt did not (Fig. 5B). These results indicate that the mutation of eIF(iso)4E-S greatly reduced susceptibility to TBTV-MW irrespective of genetic background. RT-qPCR revealed that the relative TBTV-MW accumulation was significantly lower in K326-sstt and K326-ssTT than in K326-SStt and K326-SSTT (Fig. 5C). Since there was a positive association between the ratio of plants with symptoms and virus accumulation, the reduction in the symptoms was thought to be due to suppression of virus accumulation.

Reduced susceptibility to TBTV-MW by nonsense mutation of eIF(iso)4E genes in the aphid transmission test
In general, umbravirus infects the host plant via aphids in natural environment. To evaluate the effect of the mutant eIF(iso)4E genes on TBTV-MW resistance via aphids transmission, K326-sstt and K326-ssTT, which showed reduced susceptibility in the sap inoculation test, were tested. No K326-sstt plants showed mottling symptoms even at 25 DPI, and few K326-ssTT plants showed symptoms up to 25 DPI (Fig. 6A). RT-qPCR at 25 DPI revealed significantly less accumulation of TBTV-MW in K326-sstt than in K326-SSTT, and much less in K326-ssTT than in K326-SSTT, although significant difference was not detected (Fig. 6B). The proportion of plants with symptoms was associated with virus accumulation. These results were almost similar to those in the sap inoculation test, suggesting that the mutants with sstt genotype or ssTT in eIF(iso)4E gene is effective for reduction of TBTV-MW susceptibility and eIF(iso)4E-S is one of the major factors in TBTV-MW susceptibility.

Classification of TBTV-MW
Phylogenetic analyses of viral genomic RNAs revealed that TBTV-MW is more closely related to ETBTV than to TBTV. Species demarcation criteria in the genus Umbravirus include natural host range, dsRNA pattern, nucleotide sequence identity being <70%, and little or no hybridization with cDNA probes of known viruses (Ryabov et al. 2012). Tobacco is a common natural host for both TBTV-MW and ETBTV, and a nucleotide identity of 87.7% between them is clearly higher than the criterion. Abraham et al. (2014) reported that ETBTV is distributed in the

Reduced susceptibility to a tobacco bushy top virus by eIF(iso)4Es
Breeding Science Vol. 70 No. 3 region from Ethiopia to Zimbabwe, which includes Malawi, supporting a close relationship between TBTV-MW and ETBTV. Further investigation of host plant range and dsRNA pattern may be necessary; however, these facts strongly suggest that TBTV-MW is related to ETBTV and is the causal virus of TBTD.

Involvement of eIF4E and eIF(iso)4E in TBTV-MW susceptibility
Our results showed that the suppression of eIF(iso)4E genes enabled a reduction of susceptibility to TBTV-MW. In inoculation tests, eIF(iso)4E-RNAi(+) plants showed significant reduction of susceptibility, indicating that eIF(iso)4E-S or -T, or both, is involved in susceptibility to TBTV-MW. Although we also tested for eIF4E1 and eIF4E2, a suppression of eIF4E1 did not show a reduction of susceptibility to TBTV-MW and the consistent results were not observed in eIF4E2 RNAi(+) lines. These results suggest that both eIF4E1 and eIF4E2 genes, in contrast to eIF(iso)4E, might not be involved in susceptibility to TBTV-MW. In the inoculation test with knockout mutants, mutants that had the nonsense mutation in eIF(iso)4E-S showed reduced susceptibility to TBTV-MW. This result suggests that eIF(iso)4E-S plays an important role in the life cycle of TBTV-MW. On the other hand, the eIF(iso)4E-T mutant did not show significantly reduced susceptibility. However, the numbers of symptomatic plants of sstt and K326-sstt were always less than or equal to those of ssTT and K326-ssTT in the sap inoculation test, and clearer difference between sstt and ssTT genotypes was observed in the aphid transmission test. Therefore, eIF(iso)4E-T also might be involved or partly involved in susceptibility. In Arabidopsis thaliana, the eIF4E1 knockout plants shows partial resistance to Beet western yellows virus-USA (genus polerovirus), whereas eIF(iso)4E knockout plants do not (Reinbold et al. 2013); nevertheless, the double knockout of eIF4E1 and eIF(iso)4E shows enhanced resistance (Bastet et al. 2018). Similarly, in the susceptibility to TBTV-MW mediated by eIF(iso)4E, mutant eIF(iso)4E-S works as a major resistance gene, and mutant eIF(iso)4E-T strengthens the resistance in the presence of the eIF(iso)4E-S mutation.
Resistance to potyviruses due to eIF genes is well studied. The mechanism can be explained by the interaction between VPg and the host eIF4E or eIF(iso)4E. On the other hand, certain viruses that don't have VPg use the host translation mechanism via 3ʹ cap independent translation enhancers (Truniger et al. 2017). 3ʹ cap independent translation enhancers are classified into several types (Truniger et al. 2017). Among them, the Barley yellow dwarf virus (BYDV; genus Luteovirus)-like translation element (BTE) has a characteristic 17-nt conserved sequence (5ʹ-GGAUCCUGGGAAACAGG-3ʹ) (Kraft et al. 2013, Wang et al. 2010. Both TBTV and Groundnut rosette virus have a functional BTE (Wang et al. 2010). We have confirmed that the 17-nt sequence was also conserved in TBTV-MW, possibly indicating that TBTV-MW also uses the host translation mechanism via BTE. In BYDV, BTE does not directly interact with eIF(iso)4E (Treder et al. 2008). Nevertheless, it was recently reported that resistance to BYDV was conferred by the silencing of eIF(iso)4E in wheat (Fellers et al. 2018, Rupp et al. 2019. Thus, it is still possible that eIF(iso)4E functions in translation via BTE. Considering that eIF(iso)4E forms the complex with other eIFs (Robaglia and Caranta 2006), further study for elucidating the relationship between BTE and the eIF complex is necessary. It will reveal the molecular mechanism of the reduced susceptibility to TBTV-MW caused by loss of function in eIF(iso)4E.

Use of eIF(iso)4E mutants as a genetic resource in breeding
As the double mutant showed a tendency to be more resistant than the eIF(iso)4E-S single mutant, it would be preferable to introduce both eIF(iso)4E-S and T mutations into breeding lines for TBTD resistance. To our best knowledge, the genetic resources that might show TBTD resistance have been reported only in wild Nicotiana relatives, N. clevelandii, N. acuminate, N. excelsior, and N. suaveolens (Bruin 1990, Gates 1962. Therefore, the eIF(iso)4E mutants may be the first genetic resource in N. tabacum. In addition, our study shows the first example of reduced susceptibility to an umbravirus by the suppression of eIF genes. Because of the close relation with TBTV-MW, the same mutations have a higher possibility to confer resistance to ETBTV as well. It will be important to clarify the range of resistance in the eIF(iso)4E mutants to other umbraviruses, especially ETBTV and TBTV.
The developed selection marker in the present study, which can detect the single nucleotide polymorphisms (SNPs) in both eIF(iso)4E genes, offers a useful tool for the resistance breeding. Previously, dCAPS markers for both SNPs were already designed (Takakura et al. 2018), but there was still scope for improvement in labor and cost: they required independent nested PCR and restriction enzyme digestion to detect each SNP. To solve these problems, we redesigned the system; amplification of both genes in one PCR reaction followed by one double digestion. Marker-assisted breeding for TBTD resistance will be more promoted by the newly developed dCAPS marker.
In Arabidopsis thaliana, eIF(iso)4E mutant exhibited normal growth (Lellis et al. 2002). Similarly, we observed no abnormal growth in the tobacco eIF(iso)4E mutants. However, the influence of the mutations on plant quality and yield should be evaluated in detail. For practical breeding with TBTD resistance, we have continued further studies together with investigation for strength and stability of the resistance in field conditions.

Author Contribution Statement
H.U., H.K., and Y.T. designed and planned the study. Y.T. constructed the RNAi cassette and prepared the RNAi plants. H.U. performed the viral genome sequencing, prepared the tobacco mutants, and developed the DNA marker. K.K. performed the virus inoculation test and assessed the resistance. A.S. contributed to the RT-qPCR including sample preparation. All authors interpreted the results. H.U. analyzed data, drafted the manuscript, and designed the figures. H.K. and Y.T. reviewed and edited the paper with input from all authors.