Progress 09/15/23 to 09/14/24

Outputs
Target Audience:Citrus growers and citrus industry. Scientists who are working on citrus greening, citrus plants, vector transmitted diseases and crop protection. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Ms. German, a member of the Godfrey group, received specialized training in propagating intragenic citrus through cuttings and grafting techniques. This training is designed to improve the success rate of citrus propagation. Additionally, the project manager from the Jin lab delivered a presentation to undergraduate students, demonstrating the process of creating intragenic citrus and discussing the applications of these non-GMO plants. How have the results been disseminated to communities of interest?Throughout this year, a postdoctoral researcher from the Jin lab actively disseminated our research findings to various communities of interest. She participated in several key conferences, including the International Research Conference on Huanglongbing VII (IRCHLB VII), where she engaged in discussions with colleagues about the latest technologies for generating intragenic lines and innovative methods for controlling HLB. Additionally, she attended the 2024 American Phytopathological Society meeting, where she presented our recent work on HLB control strategies to fellow researchers. Furthermore, she joined a tour at the Citrus Collection Center at UCR, where she introduced our work on the Australian finger lime to the broader citrus research community. What do you plan to do during the next reporting period to accomplish the goals?Objective. 1: Generate and examine SAMP intragenic plants that express SAMP continuously and systemically in commercially important varieties: including rootstocks Carrizo, US-942, 11 and Kuharske and scions Hamlin, Valencia, and Navel Orange Although the transformation efficiency is supper low, we are still continuously generating and testing more shoots, and screening for more positive shoots and branches, generating more cuttings. Objective 3: The CTV vector expressing SAMP will be tested on commercially important varieties for HLB resistance under the greenhouse and field conditions. Task 3.1 Inoculate commercial citrus cultivars with CTV-SAMP followed by CLas challenge The establishment of the CTV-SAMP infection (30 plants) will be confirmed using ELISA with the CTV- specific antibody (starting at two months post inoculation). The ELISA-positive trees will be examined for the presence of SAMP using Northern blot for SAMP mRNA detection as well as an immunoblot with the anti-SAMP antibody for the peptide detection (this method has been established in the Jin laboratory). The efficacy of SAMP against CLas will be assessed by challenge-inoculation of the CTV-SAMP-expressing citrus plants with the HLB-infected citrus budwood tissue or by exposing plants to CLas-containing 'hot' psyllids. The plants will be maintained and monitored under the greenhouse conditions. We can examine whether combination of intragenic rootstock and CTV will increase the accumulation of SAMP and enhance HLB resistance. The development of symptoms or the lack of those will be monitored. We will prepare 20 more plants of Valencia on US-942 rootstock, 20 more plants of Valencia on Kuharske rootstock, and 20 more plants of Navel on US-942 rootstock, and do another infection assay.. Task 3.2 Conduct a small-scale field trial to assess the efficacy of the SAMP expression from the CTV vector against CLas infection The Folimonova and Dewdney laboratories of the Co-PDs will prepare 40 'Hamlin', 'Valencia' and 'Navel' grafted on rootstock US-942. Half of them will be inoculated with CTV-SAMP and half with CTV-GFP. These plants will be tested for SAMP expression and subjected to clas infection.

Impacts
What was accomplished under these goals? Objective. 1: Generate and examine SAMP intragenic plants that express SAMP continuously and systemically in commercially important varieties: including rootstocks Carrizo, US-942, and Kuharske and scions Hamlin, Valencia, and Navel Orange. We has generated SAMP expression cassettes containing only citrus-derived DNA with phloem-specific promoter CsSUS1p and the SAMP gene from Australian finger lime. The method we used for intragenic transformation is a non-GMO biolistic bombardment method to rapidly introduce desired traits into elite varieties. We have generated SAMP intragenic rootstocks and scions in US942 and Hamlin. By screening thousands of shoots, 2 US942 plants and 1 Hamlin plant were confirmed positive for SAMP expression. We keep making cuttings from the positive plants and examining the new cutting plants. Until August 2024, we obtain 11 intragenic US942 cuttings and 6 intragenic Hamlin plants. Three positive US942 cuttings and two Hamlin grafting shoots were identified. To enhance the positive selection rate, we included a selection marker derived from the citrus genome within the SAMP expression cassette. This marker, known as PMI, is widely used as a selectable marker gene in mannose (Man) selection-based plant transformation. Until August 2024, we have 34 putative positive carrizo plants, we will do further experiments to examine the expression of SAMP. Figure 1: New cuttings from confirmed US942 intragenic plants (A). Red circles highlight three positive cuttings. The inserted SAMP cassette was detected by genomic DNA PCR (B). Figure 2: New shoots from confirmed Hamlin intragenic plants (A, B).Red circles highlight two positive shoots. The inserted SAMP cassette was detected by genomic DNA PCR (B). Figure 3:PCR Analysis on Genomic DNA to Verify SAMP Cassette Insertion in Carrizo Shoots. Objective. 2: The confirmed intragenic rootstock and scions will be tested for HLB resistance. The confirmed intragenic rootstock and scion are growing in the greenhouse. They need to be a certain size to be challenged by hot psyllids. This process requires one more year. Objective 3: The CTV vector expressing SAMP will be tested on commercially important varieties for HLB resistance under the greenhouse and field conditions. The Co-PI Folimonova lab has prepared commercial citrus cultivars - 'Valencia', and 'Navel' sweet orange plants grown on the US-942 and Kuharske rootstock. 20 plants of Valencia on US-942 rootstock, 20 plants of Valencia on Kuharske rootstock, and 20 plants of Navel on US-942 rootstock are prepared. In each set, 10 plants were grafted with 2-3 pieces of budwood from a source Citrus macrophylla plant infected with Citrus tristeza virus (CTV)-SAMP. Before grafting, we confirmed the expression of SAMP RNA in the source plant via RT-PCR. In each set, 10 other plants were grafted with budwood pieces from a Citrus macrophylla plant infected by CTV-GFP (control plants). All grafts survived and healed ensuring success of grafting. The plants were trimmed to force a new flush and systemic movement of the virus. We are monitoring these plants in our greenhouse, and they will be assayed in August for the development of CTV infection. The next step will be to perform the second round of grafting, at that time, with the budwood from a Clas-containing tree(s) (challenge inoculation). Plants have been infected with Citrus tristeza virus (CTV)-SAMP or CTV-GFP control Objective 4: Integration of research, extension, and outreach. Co-PI Vidalakis, Co-PI Dewdney, and associate Singh, the extension specialists with extensive experience, manage and direct the extension and outreach efforts in CA, FL, and LA. Drs. Jin, Huang and Folimonova and relevant personnel in their laboratories are actively involved in communicating with extension specialists and providing extension materials. Dr. Jin has been actively involved in educating the public about citrus HLB, particularly the dangers of moving plant material across state lines, through interviews with television reporters and newspaper journalists at media conferences and at LAX airport.

Publications