Abstract
The ability to express foreign genes or silence endogenous genes in plants has revolutionized both basic and applied plant biology. Systems of expression based on viruses, where the mRNA is greatly amplified by the viral replication can produce very high levels of proteins or peptides in leaves and other tissues. The citrus tristeza virus (CTV) is composed of two capsid proteins and a single-stranded RNA of about 20 kb, containing 12 ORFs and two non-translated regions called UTRs. Most CTV isolates are composed by "different" genotypes, usually with predominance of one or more sequences. Faithfully reproducing of all the genes of a ~ 20 kb RNA virus through recombinant DNA manipulations is a challenge. Such a genetic system has been developed for CTV that allows replication in protoplasts. The development of a full-length cDNA clone from the isolate T36 (CTV-T36) genome was reported in 1999. Subsequently, the development of a system for inoculation of citrus plants with recombinant CTV virus (rCTV) provided the first "pure" culture of CTV in plants. Viral genomes are efficiently released into plant cells by infiltration of tissues with cultures of Agrobacterium tumefaciens carrying binary plasmids containing full-length cDNA of viral genomes. However, attempts to agroinoculate "alemow" or "Mexican lime" with such plasmids carrying a cDNA clone of CTV-T36 were unsuccessful. Thus, a genetic system based on agroinfiltration of N. Benthamiana as an intermediate host for the production of viral particles to infect citrus plants by mechanical inoculation has been developed, and these plants were efficiently infected with viral extracts obtained from leaves of N. benthamiana agroinoculated with the cDNA clone of CTV-T36. The strategy most successful to obtain CTV vector for transient expression of a gene in the plant, was one where GPF and a controller element were added between the coat protein genes. Huanglongbing (HLB) is one of the limiting factors in the citrus industry of many countries and is caused by the bacterium Candidatus Liberibacter asiaticus (Las) having the psyllid Diaphorina citri as its vector. To date, the methods to control HLB consist in reducing the population of psyllids by insecticides and the identification and elimination of infected plants, but both having a high cost, that greatly hamper the control of this disease. So, in the long term, the most economical and sustainable strategy for the management of HLB is the production of resistant trees. Initially, the CTV vector was used to screen potential genes that could confer resistance to Las or repel the insect, with the idea of inserting such genes in plants, resulting in transgenic plants. However, the serious and devastating HLB effects in citrus plants, brought a new approach for using CTV vector by expressing anti-Las genes in the plant, as a faster alternative to control HLB compared to that one that seeks obtain transgenic plants. The great advantage of a CTV vector is that it can be transmitted by grafting to citrus varieties, avoiding juvenile characteristics and field evaluations both involved in the process of obtaining transgenic plants. The aim of this work is to obtain a CTV vector using a Brazilian strain, so it can be used for expression of genes in citrus plants that confer resistance to HLB. Its development will not only allow the transfer of technology to Brazil in the CTV building vectors but also open up new possibilities to combat HLB. Possibly, the solution for not only HLB, but for many diseases occurring in citrus plants, will come not only by using a single strategy, but rather the combination of several technologies. (AU)
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