46. Expression of a maize ribosome-inactivating protein gene in transgenic rice

Ju-Kon KIM¹, Deping XU², Xiaolan DUAN², Young-Soo HWANG¹, and Ray WU²

1) Agricultural Biotechnology Institute, Rural Development Adminisrtation, Suwon 441-707, Korea

2) Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA

Ribosome-inactivating proteins (RIPs) are plant enzymes that are remarkably potent inactivators of the eucaryotic protein synthesis system (for review, see Stirpe et al. 1992). RIPs inactivate eucaryotic ribosomes by enzymatically cleaving a specific adenine residue in the 28s ribosomal RNA. This irreversible modification renders the ribosome unable to bind elongation factor 2, thereby blocking translation. RIPS have been tested extensively for use as plant defense molecules, particularly against fungal diseases (Logemann et al. 1992; Stirpe et al. 1992). We have chosen a maize RIP gene, Zmcrip3a (Bass et al. 1992), to transform rice, aiming to produce fungal disease-resistant transgenic rice plants.

For the evaluation of the maize RIP as an anti-fungal agent in transgenic rice, we set out to use two different promoters to regulate the transgene expression, such that the RIP gene expression would be regulated in different tissues and/or at different developmental stages. In transgenic rice plants, the promoter of a rice ribulose-biphosphate caboxylase small subunit gene (rbcS) was shown to direct high-level expression of a linked reporter gene in leaf blade and sheath mesophyll cells (Kyozuka et al. 1993). Also, the 5` region of the rice actin 1 gene (Act1) promoted high-level expression of a reporter gene in virtually all tissues of transgenic rice plants (Zhang et al. 1991). The coding region of the pZmcrip3a was therefore transcriptionally fused to either the rbcS or the Act1 promoters, generating two expression vectors , pARP7 and pBY605RR, repectively (Fig. 1). The constructs pARP7 and pBY605RR were constucted based on plasmid pBY505 (B. Wang and R. Wu, unpublished) which contains the bar gene expression casstte. The two vectors also contain the bacterial bar gene used as a selectable marker in rice transformation (Fig. 1).

Rice (Oryza sativa, cv Nipponbare) suspension culture cells were bombarded with tungsten particles coated with each of the two RIP-containing plasmids, pBY605RR and pARP7, followed by selection of glufosinate ammonium-resistant

Fig. 1. Schematic representation of the plasmids pARP7 and pBY605RR. pARP7 consists of the rice Act1 5` region (1.4 kb), and the 3` region (1.0 kb) of the potato proteinase inhibitor II gene plus the bar gene expression casstte. pBY605RR consists of the rice rbcS promoter (1.3 kb), coding region of Zmcrip3a (1.0 kb)and 3` region (1.0 kb) of the potato proteinase inhibitor II gene plus the bar gene expression cassette. The bar gene expression cassette is composed of the 35S promoter (0.9 kb), bar coding region (0.6 kb), and nos 3` region (0.27 kb). Restriction sites are abreviated as follows: A, AccI; B, BamHI; Bs, BssHII; D, DraII; E, EagI; HIII, HindIII; K, KpnI; N, NotI; P, PstI; RI, EcoRI; RV, EcoRV; S, SalI; Sm, SmaI; Sp, SpeI; St, SstI; X, XhoI; Xb, XbaI; C, ClaI; G, BglII.

calli, using the procedures described previously (Cao et al. 1992). After being transferred to soil, transformed plants were obtained.

A total of 116 independent lines of plants were regenerated using the two RIP-containing constucts from 343 independent, resistant callus lines, and transferred to soil. Of these transformed lines, 56 were analyzed by genomic DNA blot hybridization to detect the integration of the transferred RIP gene into the rice genome. Total genome DNA was isolated from leaf tissues of the primary (R`0`) transformants. The DNA from plants transformed with pBY605RR was digested either with EcoRI(E), which excised the 1.0-kb RIP fragment, or with XbaI(X), which has only one site in the plasmid pBY605RR. Southern blot analysis with EcoRI-digested DNA from 27 transfromants showed hybridized bands when probed with the ³²P-labeled 0.7-kb EcoRI-HindIII fragment of the RIP gene. The results of Southern blot hybridization from 7 out of 27 Southern blot-positive transgenic lines are shown in Fig. 2. Four of the 7 lines (T1, T3, T5, and T7) appeared to contain at least one intact copy of the RIP gene, whereas three lines (T2, T4, and T6) contain rearranged copies of it. The DNA from transformants with pARP7 was similarly analyzed (data not shown). The number of integration sites or copies per genome, as determined by digesting DNA with XbaI, ranged from 1 to 10 events or copies. Transgenic lines T2, T4, T5, and T7 contain a single copy; line T1 contains 2 to 3 copies; lines T3 and T6 contain multiple copies of the RIP gene. Data of Southern blot hybridization

Fig. 2. DNA gel-blot analysis of pBY605RR-transformed transgenic rice plants. Genomic DNA isolated from the leaf tissues of T1-T7 transgenic rice plants and from an untransformed control plant (C) was digested with EcoRI(E) or XbaI(X), and hybridized with a ³²P-labeled EcoRI-HindIII-digested RIP containing restriction fragment (0.7 kb) from pBY605RR. Lane P contains EcoRI-digested plasmid pBY605RR.

Table 1. Summary of analysis of R`0` transgenic plants
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Plasmids        No. of plants    No. of Southern-   No. of Southern-positive
                  analyzed        positive plants     and fertile plants
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pARP7               25                19                      13
pBY506RR            31                27                      17
Total               56                46                      30
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analysis and the fertility scoring of these analyzed plants are summarized in Table 1. It is clearly shown that over 80% of the primary regenerated plants are Southern blot positive, and more than 50% of these plants are both Southern blot positive and fertile.

Our results demonstrate an efficient production of trnasgenic rice plants with the two expression vectors containing a maize RIP gene. Experiments are under way to examine the production of the maize RIP in the R'0' and R'1' trnasgenic plants by immuno-blot analysis. Bioassay of the transgenic rice pants for resistance to rice fungal pathogens will be initiated soon.

We are grateful to Dr R.S. Boston for sending us the maize RIP gene, pZmcrip3a, and to Baiyang Wang for providing the pBY505 plasmid. This work was supported by funds from the Rockefeller Foundation (RF93001, Allocation No.194). J.-K.K. is a Biotechnology Career Feloow of RF. Dr. X. was supported by a predoctoral fellowship from the RF.

References

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