Single amino acid substitution in the methyltransferase domain of Paprika mild 1 mottle virus replicase proteins confers the ability to overcome the high 2 temperature-dependent Hk gene-mediated resistance in Capsicum plants 3 4

1 Capsicum plants harboring the Hk gene (Hk) show resistance to Paprika mild mottle 2 virus (PaMMV) at 32 °C but not 24 °C. To identify the viral elicitor that activates the 3 Hk-mediated resistance, several chimeric viral genomes were constructed between 4 PaMMV and Tobacco mosaic virus–L. Infection patterns of these chimeric viruses in 5 Hk-harboring plants revealed responsibility of PaMMV replicase genes for activation of 6 the Hk-mediated resistance. The comparison of nucleotide sequence of replicase genes 7 between PaMMV and PaHk1, an Hk-resistance-breaking strain of PaMMV, revealed 8 that the adenine-to-uracil substitution at the nucleotide position 721 causes an amino 9 acid change from Threonine to Serine at the 241st residue in the methyltransferase 10 domain. Introduction of the A721U mutation into the replicase genes of parental 11 PaMMV overcame the Hk resistance at 32 °C. The results indicate that Hk-mediated 12 resistance is induced by PaMMV replicase proteins and that methyltransferase domain 13 has a role in this elicitation. 14 Matsumoto, 3, Virus Research The genus Tobamovirus includes devastating viral pathogens in solanaceous plants. A 1 tobamovirus, Paprika mild mottle virus (PaMMV) encodes two replicase proteins from 2 overlapping open reading frames: the 126 kDa protein and the 183 kDa read-through 3 protein (Hamada et al., 2003). The 126 kDa replicase protein contains domains with 4 methyltransferase and putative helicase activities, whereas the 183 kDa protein contains 5 an additional polymerase domain (Buck, 1999). In addition, tobamoviruses encode a 6 movement protein involved in cell-to-cell movement of the viruses and a coat protein 7 (CP) involved in the encapsidation of the viral RNA into virions. For control of 8 tobamovirus diseases, tobamovirus resistance genes, such as N gene and N’ gene in 9 tobacco plants and Tm-1 gene in tomato plants, were incorporated into commercial 10 cultivars. Virus elicitors for these tobamovirus-resistance genes differ from each other: 11 features of replicase proteins and CP are recognized by N gene-haboring tobacco plants 12 (Padgett and Beachy, 1993; Erickson et al., 1999) and Tm-1 gene-harboring tomato 13 plants (Hamamoto et al., 1997; Meshi, et al., 1988; Strasser and Pfitzner, 2007), and 14 N’gene-harboring tobacco plants (Saito et al., 1987), respectively. A common problem 15 with these tobamovirus resistance genes is that most of them lose their function at high 16 temperatures (eg. 30oC). 17 Four allelic genes at L locus, L, L, L and L, provide increased protection against 18 different kinds of tobamovirus pathotypes P0, P1, P1,2 and P1,2,3 in Capsicum plants 19 (Boukema, 1980 and 1982; Rast and Th, 1988). The tobamovirus CP is the elicitor of 20 the L genes-mediated hypersensitive response in the genus Capsicum (Berzal-Herranz et 21 al., 1995; Dardick et al., 1999; de la Cruz et al., 1997; Gilardi et al., 1998 and 2004; 22 Hamada et al., 2002; Tsuda et al. 1998). The L genes-mediated resistance also loses its 23 function at high temperatures. However, we previously identified the L gene, a new 24 allele of L genes, which confers temperature-insensitive resistance against tobamovirus 25 Matsumoto, 4, Virus Research P0 pathotype (Sawada et al., 2004). L 1a gene-mediated resistance shares a common viral 1 elicitor with temperature-sensitive L genes (Matsumoto et al., 2008). 2 In addition to the temperature-insensitive L gene, we identified a new tobamovirus 3 resistance gene in Capsicum plants, Hk (Sawada et al., 2005), which conferred 4 resistance to PaMMV but not to other tobamoviruses tested so far, including Tobacco 5 mosaic virus-Ob (TMV-Ob), Tobacco mosaic virus–L (TMV-L), Tobacco mild green 6 mosaic virus (TMGMV), and Pepper mild mottle virus (PMMoV). A remarkable feature 7 of Hk gene-mediated resistance is high temperature-dependent: it functions at high 8 temperatures such as 32°C but not at lower temperatures, (eg. 24°C), under which other 9 tobamovirus resistance genes work. The Hk gene is a single incompletely dominant 10 gene located in a chromosome differed to that of L genes. In this study we aimed to 11 identify the viral elicitor involved in the induction of Hk gene-mediated resistance in 12 Capsicum plants cultivated at high temperatures. 13 We constructed chimeric viral genomes between TMV-L and PaMMV-J, a Japanese 14 Capsicum strain of PaMMV (PaMMV-J, Hamada et al. 2003), investigated in a previous 15 study (Matsumoto et al., 2008). Chimeric tobamovirus genomes were constructed by 16 recombining DNA fragments from cDNA clones, pTLW3 (Hamamoto et al., 1997) and 17 pPAJ (Hamada et al., 2003), from which infectious virus RNA genomes are transcribed 18 in vitro. The resulting recombinant DNAs were used as templates for transcription by 19 T7 RNA polymerase (TaKaRa). RNA transcripts were mechanically inoculated onto 20 Nicotiana benthamiana with inoculation buffer (Tris-EDTA buffer, pH. 8.0 and 0.25% 21 bentonite). Infected leaves harvested 6 to 7 days post inoculation (dpi) were ground 22 with 10 mM sodium phosphate buffer (pH 7.4), and the leaf sap was used as the 23 inoculum to mechanically inoculate pepper plants cultivated in growth chambers at 24 25 °C, with a 16 h photoperiod and a light intensity of 10,000 lux. For each inoculum, 25 Matsumoto, 5, Virus Research five plants were used and experiments were performed in triplicate. Chimeric 1 tobamoviruses used in this study were named as follows: the first letter was an 2 abbreviation of the background virus (L or Pa for TMV-L or PaMMV-J, respectively), 3 followed by an abbreviation of the virus from which the recombined gene derives (L or 4 Pa), and the name of the gene (Rep, MP or CP, for replicase, movement protein or coat 5 protein, respectively). For example, L-CPPa is a TMV-L mutant whose CP gene is 6 replaced by the CP gene of PaMMV-J. 7 Virus infections in inoculated and uninoculated upper leaves at both 24 and 32 °C 8 were assessed by press blot immunoassay (Srinivasan and Tolin, 1992). In press blot 9 immunoassay, blots of inoculated leaves and uninoculated upper leaves were prepared at 10 5 and 9 dpi, respectively, and the viral CP was detected using an appropriate antibody. 11 The press blot immunoassay showed that PaMMV-J systematically infected C. annuum 12 L. cv. Nanbu-Ohnaga (Hk/Hk, Sawada et al., 2005) at 24 °C (Fig. 1); with vein necrosis 13 and systemic necrosis in inoculated leaves and uninoculated upper leaves, respectively 14 (Table 1). On the other hand, the virus induced necrotic lesions in the inoculated leaves 15 at 32 °C (Table 1) and no virions were detected in uninoculated upper leaves (Fig. 1): ie, 16 Hk gene inhibits systemic infectivity of PaMMV-J at 32 but not 24 °C. In contrast, 17 TMV-L systemically infected the Hk/Hk plants (Fig. 1) with no symptoms in inoculated 18 leaves and mosaic symptoms in uninoculated upper leaves at both 32 and 24 °C (Table 19 1). 20 All chimeric viruses caused systemic infection with mosaic symptoms in C. annuum 21 cv. Shosuke (L/L) at both 24 and 32 °C (Data not shown). When Hk plants were 22 inoculated with the chimeric viruses having the replicase genes of TMV-L (Pa-RepL, 23 L-MPPa, L-CPPa), no symptoms were induced in inoculated leaves and mosaic 24 symptoms were induced in uninoculated upper leaves at both 24 and 32 °C (Table 1). 25 Matsumoto, 6, Virus Research Furthermore, the virions were detected in both inoculated leaves and uninoculated upper 1 leaves (Fig. 1). Therefore, the chimeric viruses having the replicase genes of TMV-L 2 systematically infected Hk plants at any temperature, like TMV-L. 3 In contrast, the chimeric viruses having the replicase genes of PaMMV-J (Pa-MPL, 4 Pa-CPL and L-RepPa) failed to cause systemic infection (Fig. 1), and the viruses 5 induced necrotic lesions in the inoculated leaves at 32 °C (Table 1). On the other hand, 6 these chimeric viruses succeeded in the systemic infection of Hk plants at 24 °C (Fig. 1), 7 and vein necrosis and systemic necrotic symptoms were induced in inoculated leaves 8 and uninoculated upper leaves, respectively (Table 1). These results suggest that the 9 replicase genes of PaMMV-J are responsible for the induction of high 10 temperature-dependent Hk gene-mediated resistance to PaMMV-J. 11 A spontaneous mutant strain of PaMMV-J that overcame Hk resistance and was 12 designated as PaHk1, was isolated from a PaMMV-J-inoculated Capsicum plants 13 holding the Hk gene. The press blot immunoassay analysis showed that PaHk1 14 systemically infected Hk plants at 32 °C as well as 24 °C (Fig. 1). The chimeric viruses 15 between PaMMV-J and TMV-L showed that the replicase genes of PaMMV-J are 16 responsible for the elicitation of high temperature-dependent Hk gene-mediated 17 resistance to PaMMV-J. Therefore, we compared nucleotide sequences of replicase 18 genes between PaMMV-J and PaHk1 and found one nucleotide substitution at position 19 721 of PaHk1 replicase genes from adenine (PaMMV-J) to uracil (PaHk1). This 20 mutation causes an amino acid substitution from threonine at position 241 of replicase 21 protein to serine. The mutation resided in the methyltransferase domain. 22 To analyze the involvement of A721U in replicase genes in overcoming Hk 23 gene-mediated resistance, we constructed a mutant PaRepT241S, in which A721U 24 mutation alone was introduced. Two DNA fragments, fragment 721-1 and fragment 25 Matsumoto, 7, Virus Research 721-2, were PCR-amplified using pPAJ as a template and M4 1 (5’-GTTTTCCCAGTCACGAC-3’) and PaA789T2 (5’-GCTCCACGGAGCTTGCCTCAAG-3’), and RV 3 (5’-GTCCTTTGTCGATACTG-3’) and a primer complementary to primer PaA789T-, 4 respectively. A cDNA clone of PaRepT241S was then created by recombinant 5 PCR-amplification using fragment 721-1 and fragment 721-2 as templates and M4 and 6 RV as primers. The nucleotide sequences of recombinant DNA were analyzed using an 7 Automated DNA Sequencer Model 373 (Applied Biosystems). The mutant,