Root knot nematodes are top priority nematode pests that significantly constrain agricultural productivity globally especially in developing countries. However, expressing double stranded RNA (dsRNA) of essential nema...Root knot nematodes are top priority nematode pests that significantly constrain agricultural productivity globally especially in developing countries. However, expressing double stranded RNA (dsRNA) of essential nematode genes in susceptible plants is known to confer protection against these pests via RNA silencing. This molecular-based strategy is called host induced gene silencing (HIGS) and the selection of appropriate target nematode gene is critical to its success. In this study, therefore, we focused on root knot nematode PolA1, an essential single copy nuclear gene encoding the largest subunit of RNA polymerase I enzyme and evaluated its effectiveness as a target in conferring nematode resistance on Agrobacterium-mediated transformed tobacco plants. Transgenic tobacco expressing Meloidogyne incognita-specific (MiS) dsRNA of PolA1 gene showed significant reduction in nematode fecundity and multiplication compared to wild type plants in both T0 and T1 generations. T0 plants showed varying degrees of agronomic vigorover WT plants possibly due to varying levels of processed siRNA. However, production of MiS siRNAs in the transgenic plants coupled with significant reduction of PolA1 transcript expression in nematodes feeding on roots of transgenic plants provided evidence of HIGS. Taken together, our results show that PolA1 is a potentially effective target for HIGS-mediated reduction of root knot nematode damage on transgenic tobacco. Given the homology of our target sequence among Meloidogyne species, this protection could be broad range against other root knot nematodes aside M. incognita.展开更多
A wild rice (BKK) strain showing sterile spikelet and big leaves inhabited at the basin of the Chao Phraya river of Bangkok city, Thailand. The BKK strain was found as a natural triploid and thus its origin has been i...A wild rice (BKK) strain showing sterile spikelet and big leaves inhabited at the basin of the Chao Phraya river of Bangkok city, Thailand. The BKK strain was found as a natural triploid and thus its origin has been interested long time. Three different-sized fragments were amplified in RNA polymerase I largest subunit (<i>PolA1</i>) gene, which is a single-copy nuclear gene per haploid genome. Short type (0.14 kb) intron 20 sequence of BKK strain was identical to that of <i>O. rufipogon</i> and <i>O. sativa</i>. Phylogenetic analysis showed that long type (1.5 kb and 1.8 kb) intron 20 sequences of BKK strain were closely related to that of <i>O. longistaminata</i> and <i>O. officinalis</i>, respectively. We analyzed protein tag (Ptag) sequence encoded by exons 19 to 21 of <i>PolA1</i> gene. Determined three Ptag sequences of BKK strain were identical to that of <i>O. rufipogon</i>, <i>O. longistaminata</i>, and <i>O. officinalis</i>, respectively. Relative DNA content of nuclei in <i>O. officinalis</i> and BKK strain was 1.5 and 1.75 times than that in <i>O. sativa</i>, respectively. And BKK strain contained CentO-C1 repeats, which were unique to <i>O. officinalis</i>. These results indicated that BKK strain comprised three genomes of <i>O. rufipogon</i>, <i>O. longistaminata</i>, and <i>O. officinalis</i>.展开更多
文摘Root knot nematodes are top priority nematode pests that significantly constrain agricultural productivity globally especially in developing countries. However, expressing double stranded RNA (dsRNA) of essential nematode genes in susceptible plants is known to confer protection against these pests via RNA silencing. This molecular-based strategy is called host induced gene silencing (HIGS) and the selection of appropriate target nematode gene is critical to its success. In this study, therefore, we focused on root knot nematode PolA1, an essential single copy nuclear gene encoding the largest subunit of RNA polymerase I enzyme and evaluated its effectiveness as a target in conferring nematode resistance on Agrobacterium-mediated transformed tobacco plants. Transgenic tobacco expressing Meloidogyne incognita-specific (MiS) dsRNA of PolA1 gene showed significant reduction in nematode fecundity and multiplication compared to wild type plants in both T0 and T1 generations. T0 plants showed varying degrees of agronomic vigorover WT plants possibly due to varying levels of processed siRNA. However, production of MiS siRNAs in the transgenic plants coupled with significant reduction of PolA1 transcript expression in nematodes feeding on roots of transgenic plants provided evidence of HIGS. Taken together, our results show that PolA1 is a potentially effective target for HIGS-mediated reduction of root knot nematode damage on transgenic tobacco. Given the homology of our target sequence among Meloidogyne species, this protection could be broad range against other root knot nematodes aside M. incognita.
文摘A wild rice (BKK) strain showing sterile spikelet and big leaves inhabited at the basin of the Chao Phraya river of Bangkok city, Thailand. The BKK strain was found as a natural triploid and thus its origin has been interested long time. Three different-sized fragments were amplified in RNA polymerase I largest subunit (<i>PolA1</i>) gene, which is a single-copy nuclear gene per haploid genome. Short type (0.14 kb) intron 20 sequence of BKK strain was identical to that of <i>O. rufipogon</i> and <i>O. sativa</i>. Phylogenetic analysis showed that long type (1.5 kb and 1.8 kb) intron 20 sequences of BKK strain were closely related to that of <i>O. longistaminata</i> and <i>O. officinalis</i>, respectively. We analyzed protein tag (Ptag) sequence encoded by exons 19 to 21 of <i>PolA1</i> gene. Determined three Ptag sequences of BKK strain were identical to that of <i>O. rufipogon</i>, <i>O. longistaminata</i>, and <i>O. officinalis</i>, respectively. Relative DNA content of nuclei in <i>O. officinalis</i> and BKK strain was 1.5 and 1.75 times than that in <i>O. sativa</i>, respectively. And BKK strain contained CentO-C1 repeats, which were unique to <i>O. officinalis</i>. These results indicated that BKK strain comprised three genomes of <i>O. rufipogon</i>, <i>O. longistaminata</i>, and <i>O. officinalis</i>.
