Transgenic Tobacco Lines Expressing Yam Mosaic Virus Coat Protein-Derived dsRNA Are Resistant to Yam Mosaic Virus

Yam mosaic virus (YMV), a Potyvirus, is a highly destructive pathogen of yam accounting for yield losses up to 40%. Apart from causing significant reduction in tuber size and quality, it restricts international exchange of germplasms. It thus becomes crucial to get resistant or at least virus-free planting materials for farmers. This study was aimed at inducing resistance to YMV in tobacco by RNA silencing. An RNAi construct containing 161 bp fragment of YMV-coat protein (CP) gene was developed and used to produce transgenic tobacco lines expressing YMV-coat protein (CP) derived double stranded RNA (dsRNA) via Agrobacterium-mediated transformation. Of the eight T1 transgenic lines inoculated with YMV, six (L1, L2, L3, L5, L7 and L8) showed immunity to YMV as no symptoms were detected, whereas two (L4 and L10) exhibited high resistance with mild symptoms limited to inoculation portions. No virus could be detected in uninoculated new leaves of the transgenic lines after RT-PCR and qPCR analyses of YMV-coat protein (CP). The presence of small interfering RNAs in transgenic lines after virus challenge indicates that the resistance was acquired through RNA silencing.

Impairment of innate immune responses in cirrhotic patients and treatment by branched-chain amino acids

It has been reported that host defense responses, such as phagocytic function of neutrophils and natural killer (NK) cell activity of lymphocytes, are impaired in cirrhotic patients. This review will concentrate on the impairment of innate immune responses in decompensated cirrhotic patients and the effect of the treatment by branched-chain amino acids (BCAA) on innate immune responses. We already reported that phagocytic function of neutrophils was significantly improved by 3-mo BCAA supplementation. In addition, the changes of NK activity were also significant at 3 mo of supplementation compared with before supplementation. Also, Fisher’s ratios were reported to be significantly increased at 3 mo of BCAA supplementation compared with those before oral supplementation. Therefore, administration of BCAA could reduce the risk of bacterial and viral infection in patients with decompensated cirrhosis by restoring impaired innate immune responses of the host. In addition, it was also revealed that BCAA oral supplementation could reduce the risk of development of hepatocellular carcinoma in cirrhotic patients. The mechanisms of the effects will also be discussed in this review article.

Primary liposarcoma of gallbladder diagnosed by preoperative imagings: A case report and review of literature

A 49-year-old Japanese woman was referred to our department because of high fever and a huge abdominal mass. Computed tomography （CT） and magnetic resonance （MR） imagings revealed a tumor, about 30 cm in diameter, occupied the right hepatic lobe and the peritoneal cavity. Abdominal angiography showed that the tumor was fed mainly by the cystic artery. We preoperatively diagnosed angiosarcoma of the gallbladder and performed tumor resection with cholecystectomy because the tumor was almost casplated, however the posterior wall of the gallbladder attached to the tumor firmly. Histologically, the tumor was composed of spindle cells including lipoblasts with cellular pleomorphism, which were also detected in the muscular layer of the gallbladder. We finally diagnosed pleomorphic liposarcoma of the gallbladder. At 10 mo and 29 mo after the first operation, she underwent two more operations because of recurrence. Now she has a good quality of life 3 years and 6 mo after the first operation.

Mitigating Root Knot Nematode Propagation on Transgenic Tobacco via <i>in Planta</i>Hairpin RNA Expression of <i>Meloidogyne incognita</i>—Specific PolA1 Sequence

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.

RNAi-Mediated Resistance to Cucumber Mosaic Virus (CMV) in Genetically Engineered Tomato

Cucumber mosaic virus is one of the most constraints to the production of tomato and other vegetable crops worldwide. Here, we generated an RNAi construct containing inverted repeat of 1138 bp fragment of a partial replicase gene of CMV-O and used it to produce transgenic tomato plants expressing CMV-specific dsRNA of the replicase gene. Inoculation of transgenic plants with CMV strain O discriminated three categories of plants: plants that showed complete resistance, which were free of symptoms;highly resistant plants, which had mild symptoms, but later recovered because new leaves that emerged were free of symptoms;and susceptible plants, which showed severe symptoms similar to wild-type plants. The completely resistant lines were selected and challenged with a closely related strain, CMV-Y. Interestingly, the transgenic plant lines either remained immune or showed high levels of resistance to the strain. No virus could be detected in uninoculated new leaves of the resistant lines after RT-PCR and Dot immunobinding assay (DIBA) analyses. We could show that the resistance is correlated with post-transcriptional gene silencing because of the production of transgenic specific siRNA.

Knockdown of <i>GmSOG1</i>Compromises Drought Tolerance in Transgenic Soybean Lines

Plants are regularly exposed to myriads of stress factors that cause tremendous damage to their genetic make-up. To ensure genome stability and survival over several generations under harsher environmental conditions, plants have evolved a unique mechanism for dealing with DNA damage known as the DNA damage response pathway (DDR). It has been proposed that there may exist a relationship between the DNA damage response pathway and abiotic stress response in plants. To further investigate this relationship, we knocked down the soybean suppressor of gamma response 1 gene (GmSOG1), a master regulatory gene of the DDR, in soybean plants and subjected the generated transgenic plants to drought stress analysis. Gene expression analysis of the GmSOG1 gene in drought stressed soybean tissues revealed high levels of expression in buds and young leaves. The root lengths and root fresh weights of transgenic soybean plants grown on Murashige and Skoog media supplemented with Gamborg B5 vitamins (MSB5 media) containing 200 mM mannitol for 10 days were significantly lesser than those of drought stressed wild-type plants. Polyethylene glycol (PEG) induced drought stress assay in vivo resulted in significant damage in transgenic plants compared with wild-type plants. Also, the relative expressions of known drought responsive transcription factors such as GmDREB1 and GmLEA as well as antioxidation related genes like GmAPX and GmCAT were downregulated in transgenic soybean lines relative to wild-type plants. Moreover, wild-type soybean plants accumulated more chlorophyll and less malondialdehyde (MDA) than transgenic lines. A confirmatory experiment in GmSOG1 overexpressing Arabidopsis plants also showed significantly higher survival rates and anti-oxidation enzyme accumulation in drought stressed GmSOG1 overexpressing Arabidopsis lines compared with wild-type plants. These results suggest that the SOG1 gene may play active roles in plant abiotic stress defense.

Triploid Wild Rice (BKK) Strain Found in Bangkok Originated from Hybridizations among Three Parental <i>Oryza</i>Species

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 (PolA1) 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 O. rufipogon and O. sativa. 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 O. longistaminata and O. officinalis, respectively. We analyzed protein tag (Ptag) sequence encoded by exons 19 to 21 of PolA1 gene. Determined three Ptag sequences of BKK strain were identical to that of O. rufipogon, O. longistaminata, and O. officinalis, respectively. Relative DNA content of nuclei in O. officinalis and BKK strain was 1.5 and 1.75 times than that in O. sativa, respectively. And BKK strain contained CentO-C1 repeats, which were unique to O. officinalis. These results indicated that BKK strain comprised three genomes of O. rufipogon, O. longistaminata, and O. officinalis.