A genus of entomopathogenic Beaveria bassiana has been widely used in pest management, however little studies havebeen given to its virulence-related genes. To identify the roles of virulence genes, AtMT-induced random mutants weregenerated and followed by localization study with TAIL-PCR. Two genes, Complex I intermediated-associated protein30 (CIP30) and Autophagy protein 22 (Atg22), were predicted as virulence-related genes in B.bassiana JEF-007. To validatethe a possible relationship between two genes and fungal virulence, hpRNAi was performed. A hpRNAi plasmid wasconstructed as a model system to knock down of egfp gene in egfp-expressing B. bassiana transformant. A Real-TimePCR showed the knock down of egfp gene expression via hpRNAi. The CIP30 and Atg22 genes in B. bassiana willbe validated by the established RNAi technique. This work can be a strong platform for the functional genetics in B.bassiana.

Rice stripe virus (RSV) is one of the serious plant pathogenic viruses for rice transmitted by small brown planthopper (SBPH), Laodalphax striatellus. RNA interference (RNAi) was recently suggested as a promising strategy for controlling insect pests, including those that serve as important vectors for plant pathogens. To disturb transmission of the RSV in SBPH using RNAi pathway, we chose nine genes highly expressed in RSV-viruliferous SBPH by transcriptome sequencing. These SBPH-derived dsRNAs were applied to the insects indirectly through xylem of rice leaves by irrigation. qPCR result demonstrated that five out of nine SBPH-derived dsRNAs successfully reduced the replication of RSV in viruliferous SBPH in dose-dependent manner, suggesting that these five dsRNAs could suppress replication of RSV and provide a new tool for RSV control strategy.

담배가루이(Bemisia tabaci)는 외래해충으로 바이러스벡터로 작용하여, 토마토의 토마토황하잎말림병바이러스(TYLCV)를 비롯한 약 100여종의 바이러스를 매개하는 중요한 해충이다. 본 연구에서는 VIGS vector를 이용하여 담배가루이 방제를 위한 target 유전자들을 선발하기 위 해 gateway system을 이용한 담배가루이 cDNA library 제작을 시도하였다. 첫 번째 방법으로 oligo d(T) primer를 사용하였을 때, 평균 약 1 kb의 insert와 1.4×10 4 cfu의 titer를 확인하였다. 그러나 insert size가 너무 커서 적절하지 않았다. 두 번째 방법으로 attB-N25 random primer를 이 용하고, sonication을 6초 실시하여 다시 진행하였다. 그러나 확인되는 insert size는 다소 컸고, 몇몇은 insert가 너무 작아서 밴드가 확인 되지 않았으며, 1.04×10 5 cfu의 titer를 확인할 수 있었다. 세 번째 방법으로는 oligo d(T) primer를 이용하였고, sonication을 2초 실시하였다. 그 결 과 300 bp~600 bp size의 insert가 확인되었으나, electro transformation을 사용한 첫번째, 두번째 방법에 비해 heat shock transformation을 사용하여 titer가 5.2×10 2 cfu로 매우 낮은 것을 확인 할 수 있었다. 결과적으로 cDNA library를 만들 때 먼저 random primer를 사용하여 First strand를 합성하여 poly A를 제거하고, 다음으로 sonication을 1초 실시하여 300~700 bp정도의 적절한 size의 insert를 생성하고, 마지막으로 electro-transformation을 실시하여 transformation 효율을 높인다면 VIGS vector에 적합한 cDNA library를 만들 수 있을 것으로 사료된다.

Peptidoglycan recognition proteins (PGRPs) are family of innate immune molecules that recognize bacterial peptidoglycan. PGRP-LE, a member of the PGRP family, selectively binds to diaminopimelic acid (DAP)-type peptidoglycan to activate both the immune deficiency (IMD) and proPhenoloxidase (proPO) pathways in insects. A PGRP-LE-dependent induction of autophagy to control Listeria monocytogenes has also been reported. We identified and partially characterized a novel PGRP-LE homologue, from Tenebrio molitor and analyzed its functional role in the survival of the insect against infection by a DAP-type PGN containing intracellular pathogen, L. monocytogenes. The cDNA is comprised of an open reading frame (ORF) of 990 bp and encodes a polypeptide of 329 residues. TmPGRP-LE contains one PGRP domain, but lacks critical residues for amidase activity. Quantitative RT-PCR analysis showed a broad constitutive expression of the transcript at various stages of development spanning from larva to adult. RNAi mediated knockdown of the transcripts followed by a challenge with L. monocytogenes showed a significant reduction in survival rate of the larvae, suggesting a putative role of TmPGRP-LE in sensing and control of L. monocytogenes infections in T. molitor. These results implicate PGRP-LE as a defense protein necessary for survival of T. molitor against infection by L. monocytogenes.

RNAi 기법은 예쁜꼬마선충을 비롯하여 초파리, 생쥐, 인체및 식물들을 포함한 다양한 생물종에서 작용기작이 잘 알려져 있고, 특정 유전자들의 발현을 제어하기 위해 사용되어 왔다.본 논문은 RNAi 기법을 통해 구축된 뿌리혹선충 저항성 형질전환식물체들의 개발현황, 발전 및 응용 가능성에 대해 고찰하고자 하였다. 지난 10여년에 걸쳐 진행된 연구들을 통해 다양한 분화단계의 뿌리혹선충으로부터 분석된 79,978개의 EST가 GenBank에등록되었고, 고구마뿌리혹선충(M. incognita)과 당근뿌리혹선충(M. hapla)의 전체 게놈 염기서열이 해독됨으로써 뿌리혹선충의 기생에 관련된 단백질들 및 식물세포벽 분해 관련 효소들에 대한 정보를 이용할 수 있게 되었다. RNAi의 기본 기작은 모든 진핵생물종에서 잘 보존되어 있고, 최근 식물기생선충들에서 RNAi 효과에 대한 연구결과들이 많이 발표되었다. 서로 다른 뿌리혹선충들에서 현재까지 22종 이상의 RNAi를위한 목표 유전자들이 보고되었다. 제2령 식물기생선충(second-stage juvenile of plant parasitic nematodes)에게dsRNA의 섭취를 유도하는 octopamine, resorcinol, serotonin등의 화합물들이 발견되었고, 이를 이용하여 선충 유전자의 발현제어를 쉽게 판별할 수 있는 새로운 기술의 활용이 가능하게 되었다. 최근에 RNAi 기술의 응용 및 발전을 통해 형질전환식물체에서 발현된 dsRNA에 의한 식물기생선충 유전자들의 발현제어가 증명되었고, 이를 위한 핵심 요소들로서 적절한 선충 표적 유전자의 선택, 식물체 내 높은 함량의 dsRNA의 발현 및선충이 섭취할 수 있는 충분한 양의 dsRNA의 운반 등이 중요하다는 것이 밝혀졌다. 특히, 비표적 유전자서열(off-target gene sequence)의 발현제어를 피하기 위해 다음과 같은 사항들이 고려되어야 한다. 1)비표적 유전자서열을 확인할 수 있는 소프트웨어의 개발 및 이를 통한 비표적 유전자서열을 제거한 RNAi 벡터를 제작하여야 한다. 2)식물과 동물에서 상동성이 높은 표적 유전자의 발현을 피해야 한다. 3)전사해석틀(open reading frames)의 염기서열들 보다 상동성이 낮은 5' 혹은 3'-비해석부위(untranslated regions)로부터 표적 유전자를설계하여야 한다.

Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway is known to play a pivotal role in various cellular events and antiviral responses in both vertebrates and insects. In an attempt to elucidate the potential involvement of STAT on S. exigua-SeNPV interactions, the full length cDNA of SeSTAT was cloned from S. exigua. Analysis of temporal expression patterns shows that SeSTAT is expressed in all stages of life cycle such as larvae, pupae, and adult. Spatial expression analysis shows that it is highly expressed in fat body and Malpighian tubule. Interestingly, SeSTAT is induced at 24 h in response to either laminarin or LTA injection in larvae. Electrophoretic mobility shift assay (EMSA) shows that the binding of nuclear extracts from fat body cells immune-challenged with LTA to STAT5 probe was observed. In addition, SeSTAT was nuclear-translocalized in both fat body and gut cells that were challenged with LTA and laminarin, respectively. Finally, gene silencing of SeSTAT shows that SeNPV number appears to be increased. It suggests that SeSTAT may act as a negative regulator against SeNPV in midgut.

Small RNAs including microRNAs (miRNAs) and small interfering RNAs (siRNAs) play crucial roles in post-transcriptional gene silencing (PTGS) in eukaryotes. Small RNAs function cell-autonomously as well as non-cell-autonomously. It has been well characterized that pathogenic fungi secrete some effector molecules, which facilitate their infection into plants. However, it is not clear whether molecules in plant cells are able to move into fungal cells during infection. To test if small RNAs generated from plant cells can also move to fungal cells during infection, we generated transgenic Arabidopsis and rice plants ectopically expressing either double-stranded RNA interference (dsRNAi) or artificial miRNA (amiRNA) constructs targeting GFP gene. And then these transgenic plants were inoculated with transgenic rice blast fungus, Magnaporthe oryzae, expressing GFP transgene. Here, we showed that ectopic expression of both dsRNAi and amiRNA targeting GFP gene in transgenic plants significantly suppressed GFP expression in rice blast fungi inoculated, indicating that small RNA molecules generated in plant cells can move into infected fungal cells and efficiently degrade fungal GFP transcripts. Our results would provide a new small RNA-based strategy for the development of resistant crops against fungal pathogens.

The companion cells of the Arabidopsis thaliana egg and sperm, the central and vegetative cells, undergo active DNA demethylation prior to fertilization. However, its biological significance, extent of conservation, and targeting preferences are not yet clear. We recently showed that localized demethylation of interspersed, small transposable elements is a common feature of A. thaliana companion cells. The DEMETER DNA glycosylase encodes active DNA demethylase activity and is required for seed production. DME-mediated DNA demethylation in the central cell is required to establish imprinted gene expression in the endosperm, and is considered a master regulator for plant gene imprinting. However, the similarity among DME targets in the central and vegetative cells, despite their different functions and developmental fates, suggests that establishment of genomic imprinting may not be the basal function of DME. Lack of DEMETER in vegetative cells causes reduced methylation of transposons in sperm. Our observation suggests that the primary function of companion cell demethylation is to reinforce transposon silencing in plant gametes.

In this study, we constructed viral vector for soybean by using Soybean yellow common mosaic virus (SYCMV) infecting both Glycine max and Glycine soja. SYCMV-derived viral vector was tested to use as Virus-induced gene silencing (VIGS) vector for functional analysis of soybean genes and as protein expression vector for foreign protein expression. In vitro transcript with 5’ cap analog m7GpppG from a full-length infectious vector of SYCMV driven by T7 promoter was inoculated to soybean to test infectivity of the clone (pSYCMVT7-full). 5’-capped transcript was able to infect soybean plants. The symptoms observed in soybean plants infected by either the vector or the sap from SYCMV-infected leaves were indistinguishable, suggesting that the vector had an equal biological activity shown by virus itself. To further utilize the vector, an additional DNA-based vector was constructed. The full-length cDNA was inserted into a binary vector flanked by CaMV 35S promoter and the nopaline synthase terminator (pSYCMV35S-full). To test if the vector infects soybean and subsequently induces gene silencing, we prepared two constructs containing fragments of Phytoene desaturase (PDS) gene (pSYCMV35S-PDS1) and small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS) gene (pSYCMV35S-rbcS2) from soybean plant. Plants infiltrated with the constructs through Agrobacterium-mediated method showed distinct symptoms such as photobleaching in plants infiltrated with pSYCMV-PDS1 and pale green or yellowing in plants infiltrated with pSYCMV-rbcS2. In addition, down-regulations of mRNA levels of two genes were confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). To test if the vector can be used for foreign protein expression in soybean plants, we prepared a construct encoding amino acids 135-160 of VP1 FMDV serotype O1 Campos (O1C) (pSYCMV35S-FMDV). Plants infiltrated with the construct through Agrobacterium-mediated method showed that soybean plant infiltrated with pSYCMV35S-FMDV only was detected by Western blotting using O1C antibody. These results support that SYCMV-derived viral vector can be used as VIGS vector or protein expression vector in soybean plants.

In a model plant species Arabidopsis, one of key determinants for flowering time is a MADS-box floral repressor, FLOWERING LOCUS C (FLC). FLC is silenced after a sufficient period of winter cold has been perceived (known as vernalization response). The lack of FLC expression allows plants to achieve the competence to flower in spring through the activation of floral integrator genes. Previous studies revealed that repression of FLC by vernalization is achieved in part by an evolutionarily conserved chromatin modifying complex, Polycomb repressive complex 2 (PRC2). In Arabidopsis, PRC2 (which contains CLF, an E(z) homolog, a H3K27 methyltransferase) is recruited to FLC chromatin upon vernalizing cold and mediates methylations at Histone H3 Lys 27 (H3K27me3), a repressive histone modification mark. Recent reports identified a plethora of long and/or short noncoding RNAs (ncRNAs), which contribute to the recruitment of PRC2 to its target chromatins. In vernlaization, a long noncoding RNAs (lncRNA), named as COLDAIR, was shown to mediate FLC silencing by vernalization. COLDAIR lncRNA binds directly to CLF, a PRC2 component, and is necessary for increased enrichment of PRC2 at FLC chromatin by vernalization. In addition, we have identified additional noncoding RNAs that are involved in various developmental processes in plants. Using the FLC regulation as a model, I will discuss our current understandings on epigenetic FLC silencing by protein and noncoding RNA components. As an alternative model system, we also have studied maize endosperm development to dissect epigenetic mechanism governing its developmental processes. I will briefly discuss our progress on the understanding of maize endosperm development as well.