5.3.2
PRODUCTION OF TRANSGENIC APPLE LINES FOR SCAB RESISTANCE WITH GENES CODING FOR ONE OR TWO CHITINOLYTIC ENZYMES

JP BOLAR1, JL NORELLI1, HS ALDWINCKLE1, GE HARMAN2 and SK BROWN2

1Plant Pathology Department, and 2Horticultural Science Department, Cornell University, Geneva, NY 14456, USA

Background and objectives
Apple scab, caused by the fungus Venturia inaequalis, is the most economically important disease of apples in the north-eastern USA. The main objective of this research was to improve disease resistance of apples by using genes encoding antifungal proteins from the biocontrol agent Trichoderma harzianum. Previously we reported the production of endochitinase (ThEn-42, endo) transgenic Marshall McIntosh (MMc) apple lines with increased resistance to scab. Here the cloning and transformation of MMc apples with the gene coding for N-acetyl--D-glucosaminidase (ThNag 70, nagase) is reported. In vitro synergistic activity by endo and nagase against fungal pathogens has been demonstrated. This report details the transformation of MMc with constructs containing nagase, and endo+nagase for evaluation of in planta synergistic activity against scab.

Materials and methods
Based on sequence information of ThNag 70 [1], primers with XbaI and BamHI restriction sites were designed. A PCR-based cloning system was used to amplify the entire fragment of the gene. Two versions of the gene were obtained. For the genomic version DNA extracted from fungal hyphae of the strain P1, and for the cDNA version DNA from lambda ZAP library, were used as templates. These amplified genomic (1.9 kb) and cDNA (1.7 kb) fragments were cloned into pBluescript SK+/-, and sequenced. The ThNag 70 genes were cloned behind the double 35S promoter and AMV leader sequence of pBI525, and the entire HindIII/EcoRI cassette in pBI525 was later cloned into the binary vector pBI121. The resulting plasmids were designated as pBI121-cNag and pBI121-gNag (cDNA and genomic, respectively).

Plasmid pBIN19ESRI contains the cDNA sequence of ThEn-42 [2]. pBI121-cNag was restriction digested with HindIII and linked to adapters that had recognition sites for EcoRI+NotI+HindIII. This fragment was digested with EcoRI, and the cassette of interest was ligated into pBIN19ESRI that had been cut by the same enzyme, resulting in the double construct pBIN(endo+cNag).

An Agrobacterium-mediated transformation system was used to transfer the T-DNA of the constructs pBI121-cNag, pBI121-gNag or pBIN(endo+cNag) into MMc leaf segments. Transformation was confirmed using PCR for the presence of various fragments of the T-DNA and ELISA for detection of NPT II protein. The activity of nagase and endochitinase were tested using the fluorescent substrate 4-methylumbelliferyl N-acetyl--D-glucosaminide and 4-methylumbelliferyl--D-N,N',N"-triacetylchitotrioside, respectively.

Results and conclusions
The sequence of ThNag 70 was similar to the original published sequence. The presence of both the genes in plasmid pBIN(endo+cNag) were confirmed by PCR and restriction analysis. Total MMc transgenic lines obtained were 33, 44 and 27, for transformations with plasmids pBI121-cNag, pBI121-gNag and pBIN(endo+cNag), respectively. All lines tested positive by PCR analysis with primers specific to NPT II and ThNag 70, and ThEn-42 for lines transformed with pBIN(endo+cNag). Failure of primers specific for the vir gene to amplify DNA from transgenic lines indicated that the lines were not contaminated with Agrobacterium. Compared to the MMc control, all lines produced NPT II protein ranging from 0.01 to 0.4 ng per mg fresh weight of leaf tissue. A transformation efficiency of 3-21% was obtained as confirmed by PCR and NPT II ELISA. Nagase and endo activity were significantly greater in transgenic lines compared to the MMc control. Based on the level of enzyme production, we have selected lines that are being propagated for evaluation of scab resistance.

References
1. Peterbauer C, Lorito M, Hayes CK et al., 1996. Current Genetics 30, 325-331.
2. Hayes CK, Klemsdal S, Lorito M et al., 1994. Gene 135, 143-148.