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The project seeks to develop a simple, effective, reliable Agrobacterium tumefaciens-mediated inoculation method called agroinfection for high throughput screening sweet lines for resistance to SPLCV; prepare ihpRNA and amiRNA expression constructs for targeting SPLCV and SPFMV genes; select effective ihpRNA and amiRNA constructs to trigger resistance to SPLCV or SPFMV with Agrobacterium-mediated transient and stable transgenic expression assays in the model host plant tobacco (Nicotiana benthamiana); and transform the common sweet potato with the selected, desirable ihpRNA or amiRNA constructs, and to evaluate resistance of the transgenic lines. Outcomes/activities to be carried out include: expose undergraduate students to research and molecular biology techniques and to provide MS graduate students an opportunity to conduct thesis research related to the project under supervision of the PIs; workshops on the project will be offered to undergraduate students enrolled in the courses of plant pathology, microbiology, parasitology, immunology and plant breeding. Products generated from this project will include: <OL> <LI> An efficient agroinfection method developed in this project will be very useful to screen germplasm resistant to SPLCV in breeding program. <LI>SPLCV infectious clones generated may offer basic tools to understand plant-virus interaction and to identify viral resistant genes. <LI>The viral resistant sweet potato lines developed in this project may be released as resistant varieties or used in breeding programs.<LI>The effectively ihpRNA and amiRNA constructs created can be used to develop resistance in other sweet potato cultivars. <LI> The novel concept of ihpRNA and amiRNA technologies explored can be used to engineer durable, broad-spectrum, multiple resistances in other crops transformable. <LI> Development and application of viral resistant varieties may reduce yield losses, use of pesticides and food contamination. <LI>The establishment of a plant pathology lab will provide the essential facilities for teaching and research in seven B.S. and three M.S. programs.<LI> A lab-based plant pathology course and protocols and materials will significantly enhance ASU's teaching capacity in agricultural sciences. <LI>Availability of African-American graduates trained with hands-on skills in biotechnology will promote the viability of the economy in underserved communities. <LI>Publications in national or international journals.
NON-TECHNICAL SUMMARY: Sweet potato production is a major agricultural business in the southern U.S., valued at $283 million which represent 75% of the U.S. growing sweet potatoes. In Mississippi, sweet potato is a major horticultural crop valued at $49 million, which makes Mississippi the third in the U.S. Current cultivars are bred for fresh consumption. Efforts are being made to expand the utility of sweet potato. This will provide great opportunities for small farmers with enormous economic promise in Mississippi. However, viral infection is one of the main factors limiting the release of the full potential of sweet potato production. Viral infections can result in 30-50% yield reduction. Among sweet potato viruses in the U.S., Sweet potato leaf is considered to be the most detrimental to production. The incidence of SPLCV in the U.S. has dramatically expanded in recent years including Mississippi. SPLCV infection reportedly resulted in 25-30% yield losses to the cultivar `Beauregard' which accounts for about 80% of the U.S. production. Sweet potato viral diseases are the most difficult to control because of the lack of effective viral resistant varieties, cultural practices, and virus-killer chemicals. The advanced knowledge of the molecular mechanisms of plant-virus interactions has led to the development of novel techniques to develop cultivars that are resistant to viruses. Biotechnology provides the opportunity to translate these emerging technologies to applicable products such as viral resistant varieties. We propose to explore new technologies to develop viral resistant sweet potato varieties to address this crucial issue in Mississippi and the southern U.S. Indeed, engineering viral resistance has been achieved in papaya which has saved the U.S. papaya industry. Currently, engineered viral resistant papayas represent 53% of the U.S. production. The major impacts of the proposed project are summarized as follows: (1) the availability of viral resistant sweet potato varieties developed in this project will significantly increase its marketable yield and the income of small farmers in Mississippi and the southern U.S.; (2) it will circumvent the drawbacks, e.g., high cost and inconvenience of virus-tested `seeds' that have to be purchased annually; (3) it will make sweet potato production less dependent on pesticides which will reduce the production costs and health risks to farmers as well as to consumers; (4) the technology used in sweet potato can be explored to engineer viral resistance in other economically important crops transformable; (5) wide extension of disease resistant crop varieties will help to sustain an environmentally-friendly and sustainable agricultural system and promote responsible environmental stewardship; (6) the success of this project will enhance the reputation of ASU and will steer a wave to attract bright high school students who want to study agricultural sciences and strengthen ASU's outreach and extension programs; (7) it will enhance ASU's educational and research capacity; and (8) our positive results will benefit scientists, extension agents, and educational institutions, nationally and internationally.
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APPROACH: In the development of an inoculation method, Agrobacterial suspension of engineered A. tumefaciens bearing plasmid embracing a hemi-dimer or dimer of SPLCV genome will be prepared. The potently virulent infectious clones confirmed will be used for development of the protocol of agroinfection of sweet potato. Sweet potato will be inoculated by dipping fresh vines in bacterial suspension. After colonization, agrobacteria will inject the T-DNA including the viral DNA into plant cells where the viral replicase will be expressed, nick the two viral replication origins and ligate to form a functional circular viral genomic ssDNA. For high efficacy, bacterial suspension OD600, media, buffers, dipping time, and wounding approaches will be optimized for inoculation of sweet potatoes. To engineer broad-spectrum, highly conserved regions of all six genes of SPLCV, and six selected genes (P1, HC-Pro, CI, NIa, NIb and CP) of SPFMV will be identified from available viral genomic sequences retrieved from GenBank. The identified regions will be cloned to generate ihpRNA constructs for in planta expression. The amiRNA sequences will be selected from the conserved regions identified for ihpRNA constructs. The selected amiRNAs will be cloned by replacing the natural miRNA sequences of A. thaliana miRNA gene miR-159 or miRNA319a using overlapping extension PCR, and then transferred into a plant expression cassette of a binary vector for plant transformation. Their integrity will be confirmed by sequencing. To narrow the number of ihpRNA and amiRNA constructs and scrutinize their effectiveness, green fluorescent protein (GFP) sensor constructs will be generated. Target genes of SPLCV and SPFMV will be fused to GFP gene, cloned into a plant expression cassette of a binary vector. The expressed ihpRNAs and amiRNAs will trigger RNA silencing, knocking down the expression of the GFP-fusions of the sensor constructs. The GFP fluorescence intensity in non-infiltrated and infiltrated zones on days 3-5 post infiltration will be documented. Data will be collected over 3 days. The fluorescence will be quantified using the ImageJ program and expressed as the mean gray value. The constructs of their mean gray values that are near to positive control or less than 25% of negative control will be considered as effective constructs, be validated by detection of viral-specific siRNA or amiRNA with Northern Blot. To test their resistance, 50 plants of each transgenic line and wild type will be propagated on tissue cultures and inoculated. Their resistance will be evaluated in 50 single-plant replications by detection of the viral titers of inoculated plants with ELISA and Real-Time PCR. Transgenic lines of which their ELISA values are 3 times higher than the background or Real-Time PCR threshold cycles less than 35, will be defined as susceptible. The effective constructs confirmed in tobacco will be use to transform sweet potatoes. The yields of the resistant lines will be compared with that of wild types either infected, or non-infected with SPLCV or SPFMV or both.