No abstract

Papaya ringspot virus (PRSV) is a major limiting factor for cultivation of papaya (Carica papaya) in tropical and subtropical areas throughout the world. Although the coat protein (CP) gene of PRSV has been transferred into papaya by particle bombardment and transgenic lines with high resistance to Hawaii strains have been obtained, they are susceptible to PRSV isolates outside of Hawaii. This strain-specific resistance limits the application of the transgenic lines in other areas of the world. In this investigation, the CP gene of a local strain isolated from Taiwan, designated PRSV YK, was transferred into papaya via Agrobacterium-mediated transformation. A total of 45 putative transgenic lines were obtained and the presence of the transgene in papaya was confirmed by polymerase chain reaction amplification. When the plants of transgenic lines were challenged with PRSV YK by mechanical inoculation, they showed different levels of resistance ranging from delay of symptom development to complete immunity. Molecular analysis of nine selected lines that exhibited different levels of resistance revealed that the expression level of the transgene is negatively correlated with the degree of resistance, suggesting that the resistance is manifested by a RNA-mediated mechanism. The segregation analysis showed that the transgene in the immune line 18-0-9 has an inheritance of two dominant loci and the other four highly resistant lines have a single dominant locus. Seven selected lines were tested further for resistance to three PRSV heterologous strains that originated in Hawaii, Thailand, and Mexico. Six of the seven lines showed varying degrees of resistance to the heterologous strains, and one line, 19-0-1, was immune not only to the homologous YK strain but also to the three heterologous strains. Thus, these CP-transgenic papaya lines with broad-spectrum resistance have great potential for use in Taiwan and other geographic areas to control PRSV.

Hereditary gingival fibromatosis (HGF) is a rare, benign disorder characterized by slowly progressive fibrous overgrowth of the gingiva. To date, two loci have been mapped in familial cases with autosomal dominant non-syndromic HGF: GINGF (MIM 135300) on chromosome 2p21-p22 and GINGF2 (MIM 605544) on chromosome 5q13-q22. Of the two loci, only SOS1 (son of sevenless one, MIM 182530) gene underlying GINGF locus has been identified. Ascertainment of a large Chinese family has allowed the mapping of a novel locus to 2p22.3-p23.3, GINGF3. Haplotype construction and analysis localized the new locus to an 11.4-cM interval between markers D2S2221 (telomeric) and D2S1788 (centromeric). The maximum two-point limit of detection (LOD) score of 3.45 (theta=0) and multipoint LOD score of 5.00 for marker D2S390 strongly supported linkage to this region. Thus, this genetic interval is distal to and does not overlap with the previously described locus, GINGF, on 2p21-p22.

Melon yellow spot virus (MYSV), a tentative member of the genus Tospovirus, is considered a distinct serotype due to the lack of a serological relationship with other tospoviruses in its nucleocapsid protein (NP). Recently, a virus isolate collected from diseased watermelon in central Taiwan (MYSV-TW) was found to react with a rabbit antiserum (RAs) prepared against the NP of Watermelon silver mottle virus (WSMoV), and a monoclonal antibody (MAb) prepared against the common epitope of the NSs proteins of WSMoV-serogroup tospoviruses, but not with the WSMoV NP-specific MAb, in both enzyme-linked immunosorbent assay (ELISA) and western blotting. In this investigation, both RAs and MAb against MYSV-TW NP were produced. Results of serological tests revealed that the RAs to MYSV-TW NP reacted with the homologous antigen and the crude antigens of members of the WSMoV serogroup, including members of the formal species WSMoV and Peanut bud necrosis virus, and members of three tentative species, Watermelon bud necrosis virus, Capsicum chlorosis virus and Calla lily chlorotic spot virus. The MAb to MYSV-TW NP reacted only with the homologous antigen and the other geographic isolates of MYSV from Japan (JP) and Thailand (TH). Our results of reciprocal tests indicate that the NP and the NSs protein of MYSV are serologically related to those of WSMoV-serogroup tospoviruses. Furthermore, we show that both the MYSV NP MAb and the WSMoV NP MAb are reliable tools for identification of MYSV and WSMoV from single or mixed infection in field surveys, as verified using species-specific primers in reverse transcription-polymerase chain reaction.

Wild tomatoes (Solanum peruvianum) are important genomic resources for tomato research and breeding. Development of a foreign DNA-free CRISPR-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free CRISPR-Cas9 genome editing system based on an optimized protoplast regeneration protocol of S. peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs (siRNA) biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6) and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProSys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots. The ploidy level of these regenerants was not affected by PEG-Ca2+-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T0 regenerants and sprdr6 null T1 progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type stock and pollination with wild-type pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the wild type. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding.