Prototype Demonstration of Transgenic Resistance to the Nematode Radopholus similis Conferred on Banana by a Cystatin

Abstract

Cavendish banana was transformed using Agrobacterium tumefaciens to express a protein engineered rice cystatin (OcIdeltaD86) of value for control of plant parasitic nematodes. Expression for each line was under control of a constitutive promoter from the maize ubiquitin gene (UBI-1), a constitutive, chimeric promoter from the octopine and mannopine synthase genes of A. tumefaciens or a promoter from a root-preferentially expressed tubulin gene Arabidopsis (TUB-1). Lines were selected as of potential interest after 8 weeks challenge in containment if their mean R. similis/25 g roots for three sibling plants were more than 1 standard normal variate below the grand mean for all plants in c7-15 lines challenged concurrently. A total of 16 lines were selected on this basis as putative positives. Western blots confirmed that eight of these lines expressed cystatin with a mean of 0.08 +/- 0.04% tsp. All but two of 19 negatively selected lines from bioassays did not express cystatin. The mean resistance level of the confirmed positive lines was 69 +/- 17%. ELISA established the positive lines under control of UBI provided significantly higher expression levels of OcIdeltaD86 than recorded for the other two promoters. Lines of interest were confirmed as producing a transcript for OcIdeltaD86 by RT-PCR. Eight plants of one UBI promoter line expressing only 0.1 +/- 0.004% tsp as cystatin were re-challenged with R. similis and achieved a resistance of 70 +/- 10%. Subsequent repeat western blotting confirmed that this line still produced the cystatin after the trial. This is the first report of transgenic resistance against a major pest or disease of banana.

Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination

“…The defense function has been inferred from the ability of PhyCys to inhibit the activity of digestive proteases from arthropods in vitro, in artificial diets as well as through bioassays on transgenic plants overexpressing PhyCys genes (Pernas et al, 1998;Atkinson et al, 2004;Ribeiro et al, 2006;Alvarez-Alfageme et al, 2007). Anti-mite activities and deleterious effects against phytopathogenic fungi and viruses have also been described for these inhibitors (Gutierrez-Campos et al, 1999;Pernas et al, 2000;Martinez et al, 2003;Abraham et al, 2006;Wang et al, 2008).…”

Jasmonic acid signaling pathway of Arabidopsis thaliana is important for root-knot nematode invasion

“…Gene expression of cysteine proteinase inhibitors is closely related to the regulation of cysteine proteinase inhibitor proteins (Zhao et al, 1996). Cysteine proteinase inhibitors have been shown to affect nematode resistance, and have been used to protect various plant species including alfalfa, banana, rice, and tomato against a wide range of nematodes with diverse feeding strategies (Atkinson et al, 1996(Atkinson et al, , 2004Samac and Smigocki, 2003;Urwin et al, 1995Urwin et al, , 1997. These reports assumed repression of nematode parasitism was caused by an increase in cysteine proteinase inhibitor proteins, but our results indicated that RKN root invasion declines when cysteine proteinase inhibitor proteins increase in the plant root.…”

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

“…A number of recombinant proteins with pesticidal, antidigestive or antifeedant properties have been identified or devised over the years for the development of herbivorous pest-resistant crops by genetic transformation (Carlini and Grossi-de-Sá, 2002;Christou et al, 2006;Gatehouse, 2008;Zhu-Salzman et al, 2008). An engineered version of the rice cysteine protease inhibitor oryzacystatin I (OC-I) (Urwin et al, 1995) was shown, notably, to confer nematode resistance in transgenic banana lines genetically transformed with the corresponding transgene sequence (Atkinson et al, 2004a).…”