Development of transgenic Arabidopsis lines

4. Discussion

3.2 Development of transgenic Arabidopsis lines

Independent transgenic lines were generated which expressed hairpin RNA complementary to mRNA of COPA of T. urticae (five lines). Integration of the gene in T0 transgenic lines was confirmed by PCR (Fig. 1). The leaf disc bioassay showed all five transgenic lines of COPA to be lethal to the infested mites.

These lines were selected for detailed study. The T0 lines were self-pollinated, and seeds collected. The T1 and T2 lines were screened for glufosinateresistance to

glufosinate. All glufosinate-resistant lines showed to possess the COPA gene cassette. Lethality of T1 and T2 lines to T. urticae was evaluated (Fig. 2). Lethality to T. urticae in T0 generation, the presence of glufosinate resistance, the amplification of transgene and the lethality to T. urticae observed in the T1 and T2

generation plants indicated the stable integration of COPA gene cassette into Arabidosis genome. No apparent difference in growth and morphology was observed between the transgenic and control plants

Fig. 1. PCR results confirmed by COPA primer

(M:100 bp DNA ladder. C: Untransformed plant with same genetic background. 1-17: Expected transgenic plants for COPA transgene. Plant 3,7,8,9 and 12 are positive transgenic for COPA gene)

3.3 Lethality of Arabidopsis expressing COPA hairpin RNA against T. urticae 72 h after the infestation on Arabidopsis expressing COPA hairpin RNA (T²), a low level of T. urticae mortality (16.8 ±13.19%) was observed. The mortality

increased to 52.4 ± 11.9% and 61.7 ± 24.5% at 96 and 144 h post-infestation, respectively (Fig. 2).

Fig. 2. Toxicity test of A. thaliana expressing T. urticae COPA hairpin RNA against T.

urticae

3.4 Downregulation of COPA transcripts in T. urticae fed Arabidopsis expressing COPA hairpin RNA

qPCR analysis was performed to measure the extent of COPA gene silencing in T.

urticae fed Arabidopsis plants expressing COPA hairpin RNA. The COPA

transcript levels were reduced in T. urticae fed soybean plants expressing COPA hairpin RNA from 0 days to 6 days infestation. Compared to 0 day post-infestation (control), the COPA transcript levels were reduced 1.35-fold at 4 days post-infestation.

Fig. 2. Extent of COPA gene knockdown in T. urticae fed A. thaliana expressing T.

urticae COPA hairpin RNA

4. Discussion

Success of in planta RNAi relies primarily on the efficient expression of hairpin RNA. In current study, hairpin RNA was determined to be expressed constitutively in transgenic Arabidopsis under the control of CaMV 35S promoter.

It is unclear whether the hairpin RNA expression is tissue-specific or general in most tissues and how the expressed hairpin RNA is processed. As a substantial level of mortality of T. urticae was observed on the transgenic Arabidopsis, the expressed hairpin RNA (and/or its processed siRNA) appears to be translocated to the phloem syetem and delivered to T. urticae when sucking the phloem sap. To

maximize the delivery of expressed hairpin RNA to T. urticae, its tissue-specific expression in the sieve tube cells would be desirable, where expressed foreign transgene is directly translocated into the phloem sap, thereby facilitating the uptake of hairpin RNA into T. urticae. Since T. urticae is known to consume chloroplasts when sucking plant sap (Park and Lee 2002),, expression of target hairpin RNA in chloroplasts would also enhance the efficacy of RNAi-based T.

urticae control significantlyas recently demonstrated in the case of RNAi against

Colorado potato beetle, which was mediated by hairpin RNA expressed in chloroplasts

Selection of the target gene is of utmost importance for the effectiveness of RNAi. Since COPA is involved in the formation of coatomer-coated vesicles that are responsible for vesicular traffic between the endoplasmic reticulum and Golgi complex in eukaryotic cells. RNAi-mediated COPA knockdown may cause endomembrane system disintegration. Consequently, the RNAi-mediated down-regulation of the COPA protein may disrupt several physiological functions (e.g., autophagy, protein sorting and trafficking), thereby ultimately resulting in T.

urticae death. Nevertheless, more potent lethal gene can be still identified from

the T. urticae genome, as the initial identification of target gene (i.e., COPA) was based on a small scale screening. Since the ingestion RNAi mediated by in planta expression of lethal target gene was demonstrated to kill T. urticae, an extensive

screening of more potent lethal genes from T. urticae merits further investigation.

Since the alimentary track tissues are the first contact point to the uptaken hairpin RNA, any housekeeping genes with essential functions in these tissues would be the primary candidates.

Variation in hairpin RNA expression can occur depending on the integration site in the plant genome. Although it is not determined yet the precise location of the COPA transgene cassette in the Arabidopsis genome, it is likely to be in the intergenic region as no apparent negative aspects were noticed in the transgenic lines

In summary, our study provides a proof of concept that hairpin RNA expressed in plant hosts can induce RNAi and eventually kill T. urticae, a sap-sucking pest, as well. Our results also provide a framework for the ingestion RNAi against other sucking arthropod pests, including aphids, thrips, whiteflies, etc. Nevertheless, several questions remain to be addressed: how much target hairpin RNA was transcribed and processed to dsRNA (or siRNA), and how much of the processed RNA was translocated into the phloem sap and became available to T. urticae ingestion. Searching for more potent lethal genes and more efficient expression of hairpin RNA in plant tissues either via tissue-specific or organelle-specific transformation would be desirable.

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KOREAN ABSTRACT

흡즙성 해충인 점박이응애에 대한 섭식 RNA 간섭에 의한 치사력 검증 체계 구축

서울대학교 대학원 농생명공학부

이웅규

초록

특정유전자의 발현 조절을 위해 보편적으로 사용되는 RNA 간섭 기술은 최근 해충방제용 형질전환 식물체에 접목되어 미래의 해충 방제 전략으로 간주된다. 섭식 RNA간섭에 의한 치사력 검증 체계

구축의 일환으로서 이미 치사력이 검증된 점박이응애 유래

COPA(Coatomer subunit of coatomer protein complex) 유전자의 hairpin RNA를 일시적으로 식물체에서 발현할 수 있는 agroinfiltration 체계를 최적화하여 RNA간섭 효과를 검정하였다. Agroinfiltration의 방법에 따른 hairpin RNA 발현효율은 Carborundum을 이용한 agroinfiltration과

확인하고자 qPCR를 수행하였고, COPA의 유전자 발현이 접종 후 96시간에서 제일 많이 감소하는 것을 확인하였다.

본 실험에서는 형질전환체 개발 전 섭식 RNA간섭 체계를

구축함으로써, 짧은 시간 안에 해당유전자의 치사력을 검증할 수 있었다. 또한 모델식물인 애기장대를 대상으로 형질전환체의 치사력을 검증할 수 있었다. 앞으로 더 높은 치사력을 갖는 유전자의 선별과 hairpin RNA의 발현량을 증대시킬 수 있는 방법이 접목된다면 흡즙성 해충의 방제에 큰 기여를 할 수 있을 것으로 사료된다.

검색어: 점박이응애, Agroinfiltration, RNA 간섭, 섭식 RNAi 시스템, 학번: 2015-21787

문서에서 Establishment of test systems for evaluating ingestion RNA interference-induced lethality against Tetranychus urticae, a representative sucking pest (페이지 70-0)