Towards genetic engineering cucumber mosaic virus (CMV) resistance in lupins

Rongchang Yang

Cucumber mosaic virus (CMV) is a serious pathogen of many economically important crops. In Western Australia (WA), CMV is a serious disease of narrow-leafed lupin, Lupinus angustifolius, which is the main grain legume crop. There is no known natural resistance genes to CMV have been identified .in narrow-leafed lupin germplasm that can be transferred to new cultivars using classical breeding techniques. The aim of this project was to develop a series of molecular resistance constructs and to apply them to produce pathogen-derived resistance to CMV in narrow-leafed lupin. A total of nine different CMV resistance gene constructs were developed. Eight constructs were based on the movement protein (MP), coat protein (CP) and replicase (Rep) genes of the WA subgroup II CMV-LY isolate originally obtained from infected narrow-leafed lupin, and one was based on the CP gene of a WA subgroup I CMV isolate from banana. The gene constructs were cloned into the plant binary vectors pYR2 and pART27/7 driven by promoters from subterranean clover stunt virus (pPLEX) and cauliflower mosaic virus (CaMV 35S) and transferred into Agrobacterium (strain: AGL0). The constructs were used to transform Nicotiana benthamiana and narrow-leafed lupin with Basta as the selectable agent. For N. benthamiana a total of 1,120 explants were cocultivated with A. tumefaciens containing the pART27/7 resistance gene constructs (80 explants per construct). Following selection in culture, 16 putative transformants for each construct were transferred to the glasshouse for seed production and analysis. PCR analysis of T1 plants indicated that transformation had been successfully achieved for each of the resistance gene constructs. Transgenic plants were challenged with CMV and susceptibility or resistance was analysed by symptom development and ELISA. The results showed that some transgenic N. benthamiana plants that contained the Repm1 gene (defective CMV-LY ORF2a) were resistant to CMV-LY. In twenty-two PCR positive T₁ plants, 7 showed immunity, 12 were partially resistant, and 3 were susceptible to CMV-LY infection. In contrast, the antisense defective CMV-LY RNA 2 construct (Repm2) did not give good resistance to CMV-LY. Three of 12 T₁ plants with this construct were partially resistant (or had delayed symptoms) and the other nine were susceptible. Transgenic T1 plants containing a CMV-LY MP sense gene (MP1) showed limited resistance to CMV-LY. Two of 12 plants showed partial resistance (delayed symptoms) and two exhibited a recovery phenotype. The development of disease symptoms in the susceptible plants was faster than that in other transgenic and nontransgenic plants. Plants with the MPS transgene (untranslatable CMV-LY MP gene) showed some resistance to CMV-LY. One of 11 plants was highly resistant and three were partially resistant to CMV-LY. Three different versions of CMV-LY CP gene (CPI, CP3 and CP4) were transformed into N. benthamiana and the T₁ plants were challenged with CMV-LY. The level of resistance varied in transgenic plants depending on the CP genes present. Although a limited number of transgenic plants have been tested so far, it appears that plants containing CP4-1 show more effective resistance to CMV than transgenic plants with either CPl-1 or CP3-1. This result appears to be the first example of the use of a mutated CP gene that is longer than the wild type gene product (12 additional amino acids) and confers resistance to CMV. For narrow-leafed lupin, 12,411 explants were subjected to meristem inoculation and cocultivated with A. tumefaciens containing a replicase construct (pYRRepm1) and 3,134 explants with a movement protein construct (pYRMPS1). One hundred and sixty one independent transformants survived in vitro selection and were grafted onto compatible nontransgenic rootstocks. Fifty nine plants survived the grafting process and were transferred to the glasshouse for seed production. PCR analysis of the 59 putative transgenic lines (T0) identified 7 plants positive for the pYRRepml gene and 15 for the p YRMPS 1 gene. The complete genomic sequence of the CMV-LY isolate was also determined. The RNA1 molecule was determined to be 3,391 nucleotides (nt) in length and is predicted to contain a 5' untranslated region (UTR) of 95 nucleotides, a single open-reading frame (ORF) of 992 amino acids and a 3' UTR of 317 nt. The RNA2 molecule is 3,038 nt long and is predicted to contain a 5' UTR of 92 nt, two ORFs of 841 and 100 amino acids (ORF2a and ORF2b, respectively) and a 3' UTR of 423 nt. The RNA3 molecule is 2,003 nt long and is predicted to contain a 5' UTR of 96 nt, two ORFs of 279 and 218 amino acids (ORF3a and ORF3b, respectively) and a 3' UTR of 322 nt. Nucleotide comparisons of RNAs1-3 indicate that the LY isolate shares between 70-78% and 98-99% homology to subgroup I and subgroup II isolates, respectively. Similarly, ORFla shares 84-85% and 99% identity, ORF2a 81-84% and 94-96% identity, ORF2b 46-56% and 95-96% identity, ORF3a 82-84% and 99% identity, and ORF3b 81-83% and 99% identity. The sequence data clearly shows that there is a high degree of nucleotide and amino acid sequence homology between the CMV-LY isolate and other CMV subgroup II strains. The sequence data confirms that the LY isolate belongs to CMV subgroup II.

Towards genetic engineering cucumber mosaic virus (CMV) resistance in lupins

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