Our project is examining phloem gene expression changes in response to CLas infection in HLB-susceptible sweet orange and HLB-resistant Poncirus and Carrizo (a sweet orange – Poncirus cross). We are using a recently developed methodology for woody crops that allows gene expression profiling of phloem tissues. The method leverages a translating ribosome affinity purification strategy (called TRAP) to isolate and characterize translating mRNAs from phloem specific tissues. Our approach is unlike other gene expression profiling methods in that it only samples gene transcripts that are actively being transcribed into proteins and is thus a better representation of active cellular processes than total cellular mRNA. Sweet orange, and HLB-resistant Poncirus and Carrizo (sweet orange x Poncirus) will be transformed to express the tagged ribosomal proteins under the control of characterized phloem-specific promoters; tagged ribosomal proteins under control of the nearly ubiquitous CaMV 35S promoter will be used as a control. Transgenic plants will be exposed to CLas+ or CLas- ACP and leaves sampled 1, 2, 4, 8, and 12 weeks later. Ribosome-associated mRNA will be sequenced and analyzed to identify differentially regulated genes at each time point and between each citrus cultivar. Comparisons of susceptible and resistant phloem cell responses to CLas will identify those genes that are differentially regulated during these host responses. Identified genes will represent unique phloem specific targets for CRISPR knockout or overexpression, permitting the generation of HLB-resistant variants of major citrus cultivars.
This is the first year, 3nd quarter progress report; our grant started December 1, 2018. In the last three months, the post-doctoral researcher, Tami Collum, has started optimizing nucleic acid extraction protocols for citrus. For objective 6 (Additional Approach: Phloem limited citrus tristeza virus vectors will be used to express the His-FLAG-tagged ribosomal protein in healthy and CLas infected citrus) Dr. Dawson’s lab has all necessary constructs and has moved many of them into citrus. CTV-infected plants will soon be ready for shipment to Maryland. Again, the majority of our efforts in the 3nd quarter were focused on objective 2 (production of transgenic citrus lines). The Stover lab has performed Agrobacterium-mediated transformation of seedling epicotyls from all three citrus genotypes indicated in the grant (Carrizo, Poncirus and Hamlin sweet orange) with the His-FLAG tagged RPL18 (ribosomal protein L18) under the 35S promoter and all three phloem promoters pSUC2, pSUL and p396ss. Carrizo transgenic plants with three promoters are already acclimatized in the greenhouse: p35S::HF-RPL18 (12 plants), pSUL::HF-RPL18 (21 plants), and p396ss::HF-RPL18 (30 plants), with many plants >25 cm and suitable for taking cuttings for replication. Seven plants transformed with each promoter were evaluated for presence (PCR) and expression (RT-qPCR) of the HF-RPL18 gene, and 100% of the plants are expressing the gene. The newly transformed Carrizo with the pSUC2 promoter has been transferred to greenhouse and will be evaluated soon. Putative transgenic plants of Poncirus harboring the 35S::HF-RPL18 (12 plants) and pSUL::HF-RPL18 (10 plants) were moved to the soil. Poncirus plants with constructions p396ss::HF-RPL18 and pSUC2::HF-RPL18 are still in rooting medium (16 and 49 plantlets, respectively). Hamlin transformation was intensified in this quarter and many shoots have being transferred to rooting media, and one plant to soil. Since Hamlin has a much lower transformation efficiency, some transformations were repeated and also cotyledons have being used as a new transformation target explant for this genotype. Carrizo plants expressing the HF-RPL18 gene will be replicated and transferred to Ft. Detrick in the next quarter.
1. Please state project objectives and what work was done this quarter to address them: Objective 1. Assessing tree growth and absence of psyllids and HLB disease symptoms (including CLas bacteria titer) under protective covering (i.e., IPC). We are monitoring fruit development and retention for the second crop to be harvested next spring in the Valencia trees that were uncovered in August 2020 and had their first crop in Spring 2021. Fruit drop is starting in the non-covered HLB-infected trees. However, in the trees that were in IPC and uncovered last August, fruit drop is not happening, so far. We also found more fruit set in these trees that were uncovered last August, as compared to the non-covered trees. Fruit is also significantly larger this year, continuing the trend we observed last year.With regard to our trials with mandarin varieties, SugarBelle trees show very good growth and fruit set with no differences in growth between IPC or no-IPC conditions. Once we remove IPCs by the end of this year, we will assess fruit yield and quality as well. In contrast, Tango trees are growing significantly larger under the IPCs, as compared to non-covered trees. We will also assess fruit yield and quality in these trees when we remove the covers. Finally, Early Pride trees are not performing well under the IPCs. Typical twig dieback in this variety is exacerbated inside the covers, and trees are significantly smaller. Based on these observations, we do not recommend IPCs for Early Pride mandarin trees. Objective 2. Assessment of alternative netting approaches including targeted, alternated and patterned setup of IPC in groves for more cost-effective protection. Although HLB-positive, we are seeing lower bacterial levels (higher Ct values) in internal rows of uncovered trees planted in an alternate pattern, which suggests that internal rows in a grove may have some protection if external tree rows are covered by IPCs. These studies need to be continued and refined to clearly determine if we can take advantage of the ‘cross-protection’ as well as the edge effect. Objective 3. Monitoring the transition from vegetative to reproductive stage in the covered and non-covered trees. As stated in our last quarterly report and in Objective 1 of this report, we are assessing fruit development, and did not find significant differences in fruit set as compared to non-covered trees, but fruits inside IPCs seem to be larger. This is promising, since IPC protection could potentially be prolonged to get the trees well into the productive age, producing high quality fruit, as we have shown in our last report. By applying brassinosteroids as a combined treatment with IPCs we expect to prolong tree health further, and produce a commercial-size good quality fruit crop. Objective 4. Comparing IPC with CUPS-like systems. We are now monitoring fruit growth inside the CUPS to later compare with IPCs. We are also monitoring fruit drop and are ready to start with quality assessment in Tango, as fruit growth seems to be well advanced inside the CUPS and ready to reach commercial maturity. Outreach for this quarter:-Alferez, F., Albrecht, U, Gaire, S., Batuman, O., Qureshi, J., Zekri, M. Individual Protective Covers (IPCs) for young tree protection from the HLB vector, the Asian citrus psyllid. EDIS, accepted, in press. -Alferez, F, Batuman, O, Gaire, S, Albrecht, U, Qureshi, J. Assessing spatial patterns of IPCs deployment in young citrus. Citrus Industry, August, 2021. -Gaire, S, Alferez, F, Albrecht, U. Horticultural attributes of SugarBelle, Tango and Early Pride mandarin trees grafted on two different rootstocks grown with and without individual protective covers (IPCs). ASHS Annual meeting. August 5-9, 2021, Denver CO. -Alferez, F and Batuman, O. Individual Protective Covers (IPCs) and their patterned use for young tree protection. CRAFT Growers meeting, August, 2021 2. Please state what work is anticipated for next quarter: Next quarter will be the final quarter of this project. Although we anticipate finishing most of the work in this project, some of our results warrant continued research that can be of great interest in providing real guidance on what can be done to keep trees healthy at least for several years into their productive age.Objective 1. We will continue with regular work pertaining horticultural/pathological parameters in all plots. We will remove the covers in the mandarin trials and will assess fruit yield and quality. When new funding become available, we plan to start brassinosteroids treatments, as we already know that this treatment can prolong health of citrus trees by delaying HLB infection and reducing incidence of other pests and diseases, which potentially can result in better fruit yield and quality at least in the mid-term. The real positive impact of this combined treatment, and for how long can we benefit from it, remains to be determined.Objective 2. We will continue collecting data on psyllid population and HLB incidence in the different netting layouts (i.e., pattern). We will confirm if the trend that we observed in Ct values is maintained or changes over time.Objectives 3 and 4. We will continue collecting data on fruit growth and maturation for the second season of deficit irrigation treatments. We will also monitor for early fruit drop if occurs, and will assess fruit quality and yield. 3. Please state budget status (underspend or overspend, and why): We continue on track with activities and spending. Budgeted amounts for salaries are being spent as predicted.
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