The protein-protein interaction of virus and host is essential for virus infection and host defense. The coat protein (CP) of tomato mosaic virus (ToMV) has been proved to be involved in cell-to-cell and long-distance movements of viruses that are presumably related with the protein-protein interactions. However, the host proteins that interact with the ToMV coat protein (ToCP) are largely unknown. In this study, we isolated a cDNA from a tobacco library through yeast two-hybrid system, which encodes a protein designated the ToMV CP-interacting protein-L (IP-L) that interacted with ToCP in vitro and in vivo. Sequencing analysis revealed that the putative coding region of IP-L gene was identical to that of an 'elicitor responsive protein' gene from N. tabacum (Genbank: #AB040409). A homology was also found between the cDNA sequence of IP-L and two senescence-related cDNAs (SENU1: Z75523 and AY479987) isolated from tomato and pepper. Northern blotting analysis showed that the mRNA level of IP-L was elevated after infection of ToMV. Then, we investigated the in vivo function of IP-L using virus-induced gene silencing (VIGS) and virus challenging assay. Semi-quantitative RT-PCR and Northern blotting results showed that the endogenous mRNA of IP-L in N. benthamiana plant was silenced at 10 days post inoculation with the in vitro transcripts of PVX-IP-L that were produced from the potato virus X (PVX)-based gene silencing plasmid pPC2S.IP-L. The IP-L silent plant developed a delayed systemic symptom at 7 days post challenging with ToMV, indicating that a high expression of IP-L was necessary for the interaction with ToCP to assist the viral transportation. Together, our data suggested that IP-L is a novel plant factor that interacts with the coat protein of ToMV and facilitates the long-distance movement of virus, which may provide a valuable clue for us to further investigate the mechanisms of plant virus infection and to control plant virus diseases.

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The growth area of living modified (LM) cotton has steadily increased every year, since its first commercialization in 1996. Development of environmental risk assessment tools and techniques for LM cotton is required for ecosystem safety. We therefore developed multiplex PCR assays for simultaneous detection of two (MON15985, MON531) and four (GHB614, LLCOTTON25, MON88913 and MON1445) LM cotton events approved in Korea, with event specific primer pairs. The PCR reactions were optimized by using event specific primers of six LM cottons at various concentrations. The reactions allows amplification of estimated amplicons of MON15985 (214 bp), MON531 (270 bp), GHB614 (119 bp), LLCOTTON25 (164 bp), MON88913 (276 bp), and MON1445 (389 bp) from multiplex PCR reactions. The multiplex PCR assay developed allowed that two annealing steps (15 cycles at 55°C and 25 cycles at 60°C) were performed for amplification of distinguished two LM cottons, and only one annealing step (50 cycles at 60°C) was necessary for tetraplex PCR. Primer extension step of all PCR reactions was skipped for time-effective amplification. Our methods suggest that two multiplex PCR assays can be cost-effective and a rapid diagnostic tool for environmental LMO monitoring of six LM cottons.

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