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Science in China Ser. C Life Sciences 2004 Vol.47 No.4 382—388

Effect of cDNA fragments in different length derived from Potato Virus Y coat protein gene on the induction of RNAmediated virus resistance ZHU Junhua, ZHU Xiaoping, WEN Fujiang, BAI Qingrong, ZHU Changxiang & SONG Yunzhi Laboratory of Plant Genetic Engineering, College of Plant Protection, Shandong Agricultural University, Taian 271018, China Correspondence should be addressed to Wen Fujiang (email: [email protected])

Received August 18, 2003

Abstract We have reported that cDNA derived from entire coat protein (CP) gene of potato virus Y (PVY) could induce resistance to PVY infection in transgenic tobacco plants, and the resistance was further demonstrated to be RNA-mediated rather than coat protein-mediated. In this study, we cloned cDNA fragments of 202 bp, 417 bp, and 603 bp in length derived from the 3′end of the PVY CP gene, and the cDNA fragments were introduced into tobacco (var. NC89) plants via Agrobacterium-mediated transformation system. The results of resistance assay showed that the CP cDNA fragments of 417 bp and 603 bp could confer resistance of the transgenic plants to PVY infection, but the fragment of 202 bp in length could not. Molecular analysis revealed that the resistance was RNA-mediated, which is believed to be a result of post-transcriptional gene silencing. The results indicate that the length of cDNA fragments needed for resistance induction was located somewhere between 202 bp and 417 bp from the 3′ end of PVY CP gene. Keywords: PVY, partial length of CP gene fragments, transgenic tobacco plants, RNA-mediated resistance, post-transcriptional gene silencing. DOI: 10.1360/03yc0066

Potato virus Y (PVY) is a main viral pathogen infecting economic crops such as potato and tobacco plants. Genetic engineering has been so far the most effective method to produce viral resistant plants. Because of the shortage of viral resistant genes in plants, cDNAs derived from viral genes were often used for induction of resistance in transgenic plants (the socalled “pathogen-derived resistance”)[1]. Among the genes used in the pathogen-derived resistance strategy, the coat protein genes of viruses were most frequently — used[2 6]. Earlier studies implied that the resistance to viral infection in the transgenic plants could be conCopyright by Science in China Press 2004

ferred by the CP protein, i.e. the resistance could be protein-mediated [7]. Later, more studies demonstrated that, instead of the CP protein, the resistance could be RNA-mediated. Recent studies reveal that the RNAmediated viral resistance is very similar to post-transcriptional silencing (PTGS)[6,8,9]. In most reported studies on RNA-mediated viral resistance, entire genes (cDNAs) were frequently used as transgenes, though fragments of partial genes (cDNAs) were also used in some cases in which the induction of RNA-mediated resistance was proved to

Different length on RNA-mediated virus resistance

be dependent on the length of transgene frag— ments[10 13]. Determination of the effective lengths of specific genes of viruses needed for resistance induction in transgenic plants might be helpful in production of transgenic plants against multi-viral infections, and it also could be beneficial in understanding the mechanism of RNA-mediated viral resistance or PTGS. However, little is known at present about the influence of transgene length on the induction of RNA-mediated resistance against important viruses such as PVY. Our previous studies demonstrated that the entire cDNA of PVY CP gene could induce viral resistance in transgenic tobacco plants, and the resistance was further proved to be based on the transgene RNA (RNA-mediated resistance)[6]. In this work, we used cDNA fragments of 202 bp, 417 bp, and 603 bp in length derived from the 3′end of the PVY CP gene as transgenes, and the cDNA fragments were introduced into tobacco plants via Agrobacterium-mediated transformation system. Resistance assay demonstrated that the effective length of CP fragments for resistance induction might be located somewhere between 202 bp and 417 bp from the 3′end of PVY CP gene. 1 1.1

conducted by PCR using a construct containing cDNA representing the whole length of CP gene (804 bp) constructed previously[6], and the fragments were cloned into pBSK plasmid, obtaining pBSK-CP202, pBSK-CP417, and pBSK-CP603, respectively. Primers were designed as follows. Primers for the 1/4 CP: 5′ -GCGCGGATCCCTTTCATGAGGTCACATCACGAAC-3′ (upstream), 5′-GCGCGGTACCTCACATGTTCTTGACTCCAAGTAC-3′(downstream); primers for the 1/2 CP: 5′ -GCGCGGATCCGGAGTTTGGGTTATGATGGATG-3 ′ (upstream), the downstream primer was the same as that used in the 1/4 CP cloning; primers for the 3/4 CP: 5′-GCGCGGATCCGAACACTTGCTCGAGTATGCTC-3 ′ (upstream), and the downstream primer was the same as that used in the 1/4 CP cloning. BamH I and Kpn I restriction sites were introduced into upstream and downstream primers, respectively (underlined bases). All the fragments contained no translation start codons in the reading frames. The location of the three cDNA fragments in the CP gene is illustrated in fig. 1.

Materials and methods Materials

Necrosis strain of PVY (PVYN) was used in this study, which was kindly provided by Dr. Liu Dehu of the Chinese Academy of Sciences. Escherichia coli (E. coli) strain DH5α, Agrobacterium tumefaciens strain LBA4404, DNA vector of pROKII, and antiserum of PVYN were maintained in the laboratory. Nicotiana tabacum L. var. NC89 was used in transformation. Taq DNA polymerase, restriction endonucleases and T4 DNA ligase were obtained from TaKaRa Company. Probe labeling kit and HybondTM-N+ were provided by Promega and Amersham Pharmacia, respectively. 1.2

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Methods

(1) Cloning of cDNA fragments from partial CP gene of PVYN. Three cDNA fragments were cloned from the 3′end of PVYN CP gene, i.e., fragments of 202 bp (1/4 CP), 417 bp (1/2 CP), and 603 bp (3/4 CP) in length at the 3′end of the CP gene. Cloning was

Fig. 1. Locations of the cDNA fragments of CP202, CP417 and CP603 in PVY CP gene.

(2) Construction of plant expression vectors. The cDNA fragments were obtained by digesting the pBSK-CP202, pBSK-CP417, and pBSK-CP603. The cDNA fragments were digested with BamH I and Kpn I followed by isolation from low-melting agarose gel. The fragments were then inserted into binary vector pROKII under the driving of CaMV 35S promoter and under the control of nos terminator. The plant expression vectors obtained were termed as pROKII-CP202, pROKII-CP417, and pROKII-CP603, respectively. (3) Plant transformation and analysis of the transgenic plants. The plant expression vectors were introduced into Agrobacterium tumefaciens LBA4404 via frozen-throwing method, and eventually introduced into N. tabacum L. var. NC89[14]. Transgenic

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plantlets were obtained under the selection pressure of Kanamycin. The transgenic plants were further confirmed using PCR, Dot-blot hybridization, and the 2nd round of Kanamycin selection. (4) Viral resistance assay of the transgenic plants. The virus inoculum was prepared by grinding N PVY -infected leaves, and diluting with phosphate buffer at a ratio of 1︰10 (w/v). The leaf extract was used to inoculate transgenic plants at the growth stage of the 4th or 5th leaf. The resistance was detected by both symptom observation and ELISA. (5) Western blot analysis. Western blot analysis was conducted as described previously[6]. (6) Southern blot analysis. Total plant DNAs of both transformed and untransformed plants were isolated using the method of hexadecyltrimethylammonium bromide (CTAB). The total DNA was digested with Hind III and used for Southern analysis as described previously[6]. (7) Northern blot analysis. Total plant RNA was isolated using the method of guanidine thiocyanate-phenyl-chloroform[15]. RNA was dissolved in RNase-free distilled water treated with DEPC. 10 μg of total RNA was denatured and fractionated by electrophoresis on 1.2% (w/v) agarose gels containing formaldehyde. The RNA was blotted onto HybondTM-N+ membranes with 20×SSC and hybridized for 24 h at 65℃ with specific α32P-labelled probes. 2

Results

2.1 Cloning of the cDNA fragments, construction of expression vectors, and production of the transgenic plants cDNA fragments of CP202, CP417, and CP603 were obtained by PCR using the PVYN-CP as template DNA. The fragments were then inserted into pBSK plasmid, and amplified in DH5α. The sequences of the three cDNA fragments were confirmed as the sequences expected by DNA sequencing (TaKaRa Company). Plant expression vectors containing the three cDNA fragments were obtained by inserting the cDNAs into binary vector of pROKII. Bacterium and

Science in China Ser. C Life Sciences

plant transformations were done as described in Section 1.2. Transgenic plants were screened by Kanamycin selection, PCR and Dot blot hybridization. As a result, 256, 224, and 77 transgenic plants (To) were obtained from transformation using pROKII-CP202, pROKII-CP417, and pROKII-CP603, respectively. 2.2

Resistance assay against PVY infection

Tobacco plants were inoculated with PVY-infected leaf extract by mechanical inoculation. About one week after-inoculation, all the untransformed plants and plants transformed with pROKII plasmid (control plants) showed vein-clearing symptom which later developed into mosaic and vein-necrosis symptoms. For the plants transformed with CP202, CP417, and CP603 fragments, three types of reaction to PVY infection were observed. In type I, transgenic plants got as sick as control plants (susceptible or S type); in type II, the symptom and virus concentration (as determined by ELISA) were delayed about 3 days compared to control plants (delayed or D type); in type III, the transgenic plants were immune to virus infection, no symptoms or virus were detected during the entire life time of the plants (resistant or R type) (table 1). In order to exclude false resistant reactions in the R type plants, re-inoculation of the R-plants was conducted 15 days after the first inoculation. Table 2 summarizes the results of resistance assay of the transgenic plants. Of the 256 plants transformed with CP202, no delayed and resistant types of plants were observed; of the 224 plants transformed with CP417, 24 plants (10.71%) showed delayed type, and 19 plants (8.84%) turned out to be R type; and of the 77 plants transformed with CP603, 6 plants (7.79%) showed delayed symptoms, and 8 plants (10.39%) showed R type reaction. 2.3

CP protein detection in the transgenic plants

To determine if the transgenes (cDNA fragments) were translated in the transgenic plants, Western blot and ELISA were used in CP detection. The results showed that no CP was detected in all the transgenic plants (data not shown), indicating that the transcripts of the transgenes were not translated.

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Table 1 ELISA results of some transgenic plants with CP202, CP417 and CP603 Transgenes CP202

Transgenic plants Symptom 7 dpi 15 dpi 30 dpi 45 dpi CP202 (2) S 0.498 ± 0.008 0.911 ± 0.008 0.935 ± 0.014 0.933 ± 0.014 CP202 (46) S 0.468 ± 0.009 0.876 ± 0.011 0.891 ± 0.006 0.886 ± 0.019 CP202 (123) S 0.502 ± 0.002 0.942 ± 0.015 0.952 ± 0.008 0.974 ± 0.008 CP202 (210) S 0.474 ± 0.011 0.906 ± 0.007 0.911 ± 0.004 0.927 ± 0.017 CP202 (224) S 0.412 ± 0.006 0.892 ± 0.009 0.891 ± 0.008 0.898 ± 0.028 CP417 CP417 (6) S 0.486 ± 0.005 0.841 ± 0.005 0.872 ± 0.006 0.880 ± 0.014 CP417 (11) S 0.543 ± 0.008 0.986 ± 0.006 0.994 ± 0.005 0.914 ± 0.007 CP417 (23) S 0.452 ± 0.006 0.942 ± 0.007 0.948 ± 0.005 0.962 ± 0.019 CP417 (74) S 0.472 ± 0.007 0.884 ± 0.011 0.882 ± 0.009 0.872 ± 0.014 CP417 (81) S 0.561 ± 0.008 0.905 ± 0.012 0.924 ± 0.008 0.957 ± 0.014 CP417 (1) D 0.123 ± 0.005 0.485 ± 0.009 0.868 ± 0.008 0.854 ± 0.004 CP417 (35) D 0.134 ± 0.006 0.504 ± 0.004 0.902 ± 0.013 0.898 ± 0.005 CP417 (89) D 0.098 ± 0.012 0.482 ± 0.015 0.888 ± 0.008 0.864 ± 0.012 CP417 (105) D 0.185 ± 0.009 0.527 ± 0.015 0.987 ± 0.015 0.989 ± 0.006 CP417 (169) D 0.084 ± 0.015 0.468 ± 0.005 0.842 ± 0.009 0.827 ± 0.008 CP417 (59) R 0.069 ± 0.008 0.072 ± 0.005 0.075 ± 0.016 0.075 ± 0.004 CP417 (77) R 0.057 ± 0.012 0.058 ± 0.005 0.062 ± 0.024 0.064 ± 0.006 CP417 (95) R 0.084 ± 0.015 0.075 ± 0.008 0.084 ± 0.009 0.086 ± 0.007 CP417 (157) R 0.067 ± 0.006 0.078 ± 0.004 0.082 ± 0.007 0.074 ± 0.018 CP417 (202) R 0.058 ± 0.006 0.059 ± 0.006 0.064 ± 0.009 0.056 ± 0.008 CP603 CP603 (39) S 0.456 ± 0.005 0.808 ± 0.005 0.822 ± 0.008 0.824 ± 0.018 CP603 (59) S 0.520 ± 0.004 0.828 ± 0.003 0.844 ± 0.012 0.868 ± 0.008 CP603 (126) S 0.488 ± 0.005 0.831 ± 0.008 0.838 ± 0.012 0.845 ± 0.018 CP603 (166) S 0.469 ± 0.008 0.795 ± 0.009 0.809 ± 0.004 0.824 ± 0.018 CP603 (181) S 0.548 ± 0.009 0.889 ± 0.004 0.924 ± 0.008 0.922 ± 0.004 CP603 (36) D 0.104 ± 0.004 0.521 ± 0.024 0.877 ± 0.008 0.896 ± 0.019 CP603 (42) D 0.084 ± 0.006 0.485 ± 0.009 0.822 ± 0.012 0.842 ± 0.014 CP603 (56) D 0.098 ± 0.008 0.466 ± 0.007 0.798 ± 0.017 0.821 ± 0.028 CP603 (154) D 0.109 ± 0.008 0.511 ± 0.018 0.974 ± 0.016 0.968 ± 0.012 CP603 (167) D 0.068 ± 0.009 0.475 ± 0.004 0.877 ± 0.004 0.886 ± 0.015 CP603 (100) R 0.096 ± 0.007 0.085 ± 0.008 0.084 ± 0.015 0.086 ± 0.025 CP603 (121) R 0.085 ± 0.008 0.078 ± 0.006 0.078 ± 0.024 0.082 ± 0.014 CP603 (173) R 0.084 ± 0.006 0.086 ± 0.005 0.081 ± 0.008 0.079 ± 0.007 CP603 (188) R 0.067 ± 0.005 0.085 ± 0.005 0.084 ± 0.016 0.075 ± 0.015 CP603 (182) R 0.083 ± 0.007 0.112 ± 0.004 0.095 ± 0.015 0.082 ± 0.025 +CK1 S 0.614 ± 0.007 0.922 ± 0.014 － － − pROK II +CK2 S 0.596 ± 0.006 0.854 ± 0.008 － － 0.052 ± 0.005 － － － −CK1 − pROK II 0.048 ± 0.005 － － － −CK2 The values in the table are the average ELISA readings with standard deviations of four times. +CK1, Untransformed tobacco (NC89) inoculated with PVY; +CK 2, pROK II-transformed tobacco inoculated with PVY; −CK1, untransformed tobacco (NC89) without PVY inoculation; −CK2, pROK II-transformed tobacco without PVY inoculation; S, susceptible; D, delayed; R, resistant; dpi, days post inoculation.

Table 2 Responses of transgenic plants expressing CP202, CP417 and CP603 against PVY infection Transgene CP202 CP417 CP603 pROKII (pROKII-transformed tobacco) − (untransformed tobacco)

Total number of transgenic plants 256 224 77 42 30

Number of susceptible plants 256 181 63 42 30

Number of delayed plants 0 24 6 0 0

Number of resistant plants 0 19 8 0 0

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2.4

Science in China Ser. C Life Sciences

Southern blot analysis

Total DNAs isolated from R and S transgenic plants were digested with Hind III, and then subjected to Southern blot using PVYN-CP as probe DNA. Fig. 2 shows some of the typical Southern blotting results. Hybridization bands were observed in the lanes containing DNAs from CP202-, CP417-, and CP603transformed plants, but not in the lanes containing control DNA (DNA from untransformed or pROKIItransformed plants), demonstrating that the plants Were genetically transformed. Although the number of DNA bands (insertions) varied for different types of transgenic plants, no clear relationship between resis-

tance induction and transgene copy (insertion) number was observed. 2.5

Northern blot analysis

Total RNA was isolated from some of the transgenic plants, and then used in Northern blot analysis (fig. 3). The accumulation of transgene transcripts varied among the plants with different reaction types. The transgene RNA accumulated less in R type plants than it did in plants of S type, i.e., there was a negative relationship between resistance and the amount of RNA accumulation in the transgenic plants, which was coincident with our previous conclusions[6].

Fig. 2. Southern blot analysis of genomic DNA from transgenic plants. Genomic DNAs from resistant (R) and susceptible (S) plants transformed with CP202-, CP417-, and CP603 were digested with Hind III, separated by electrophoresis, blotted to HybondTM-N+membrane and hybridized with PVYN-CP probe. The number of bands implied copy numbers or insertion numbers. NC89, Untransformed tobacco (NC89); pROK II, pROK IItransformed tobacco. The numbers under S and R are the serial numbers of transgenic plants.

Fig. 3. Northern blot analysis of the transgenic plants. (a) Result of Northern blot; (b) equalized RNA in each lane (10 μg). RNA extracted from resistant (R) and susceptible (S) plants transformed with CP202-, CP417-, and CP603 was subjected to denaturing gel electrophoresis, blotted to HybondTM-N+membrane and hybridized with a radioactively labeled PVYN-CP probe. NC89, Nontransformed tobacco (NC89); pROK II, pROK IItransformed tobacco. The Numbers under S (R) are the serial number of transgenic plant.

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3

Discussion

RNA-mediated resistance is a new strategy of genetic engineering against virus infection in plants, which usually confers extreme resistance (immunity) against virus infection[16]. RNA- mediated resistance is also thought to be safer than protein-mediated resistance, for only part of a virus gene is needed for resistance induction in the transgenic plants. Because of this characteristic, it could be easier to produce transgenic plants resistant to multi-virus infections by constructing short fragments of genes from different viruses into a single chimeric transgene[17]. Therefore, determination of the shortest effective fragment of a given viral gene is critical for this purpose. It had been shown in a few reports that gene fragments from the 3′end of a gene might be more effective in inducing resistance or PTGS[12,18~20]. For simplicity, we used cDNA fragments with various lengths in the 3′end of PVYN-CP gene. The results demonstrated that transgenic plants containing 1/2 CP (417 bp) and 3/4 CP (603 bp) could effectively develop viral resistance against PVY infection, whereas the plants transformed with the 3/4 CP (202 bp) could not. This result indicates that the shortest effective length of the CP gene fragment could be located somewhere between 202 bp and 417 bp from the 3′ end of the CP gene. In the few reports so far about the studies on the induction of viral resistance or PTGS by gene fragments, most researchers connected the short DNA fragment with another DNA fragment called “silencer DNA”. Therefore, the actual transgenes were bigger than the DNA fragments needed for specific resistance or PTGS induction. Sijen et al. demonstrated that a fragment with 60 bp in length of movement protein gene of Cowpea mosaic virus (CpMV) was enough to initiate virus resistance against CpMV in transgenic plants if the fragment was linked with potato virus X (PVX)[12]. Thomas et al. linked DNA fragments of green florescence protein (GFP) gene with PVX, and demonstrated that only a fragment of 28 bp of the GFP gene was enough to induce PTGS targeting specifically the GFP transcripts in plants transformed with

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GFP gene[21]. In these studies, only a few used DNA fragments derived from a virus gene directly as transgenes[13,20]. Pang et al. introduced DNA fragments in various lengths derived from the nucleocapsid protein (N) gene of Tomato spotted wilt virus (TSWV) into plants for viral resistance induction. When the fragment was linked with a silencer DNA (GFP gene), a short fragment of 110 bp in length could induce viral resistance in transgenic plants, whereas the short fragment alone could not induce any resistance. A fragment about half of the TSWV-N gene (about 380 bp) was needed for resistance induction. Why is there a limit on the shortest length of transgene for virus resistance or PTGS induction? The exact reason for this is still unknown. It has been believed that PTGS is triggered by double stranded RNA (dsRNA) molecules that could be produced from either aberrant RNA (aRNA) or small interfering RNAs (siRNAs)[10,22]. In recent models describing the molecular mechanism of PTGS, dsRNA production is believed to be a critical step for PTGS initiation[23,24]. The siRNAs could also act as signals for PTGS transduction in the silenced plants[25,26]. It is believed that RNA-dependent RNA polymerase (RdRP) might be involved in dsRNA synthesis from the aRNAs[10,27]. When the transgene is too short, it might be difficult to produce aRNAs or siRNA, therefore lacking the dsRNA for PTGS induction. Anyway, more studies are needed to understand this question. There is still controversy for whether transgene copy number affects the induction of PTGS or not. In some studies, the results indicated that there was a positive relationship between PTGS induction and transgene copy numbers[6,28], but in other studies, no such a relationship was observed[29]. The way of transgene incorporation might be more important than the copy number for PTGS induction. Acknowledgements We would like to express our thanks to Drs. Fang Rongxiang, Chen Xiaoying and Pang Yongqi for their technical help in molecular analysis of the transgenic plants in this research. This work was supported by the National Natural Science Foundation of China (Grant No. 30270875), Shandong Province Natural Science Foundation (Grant No. Z2000D02), and Shandong Province Science and Technology Development Project.

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