UNDERSTANDING HOST-PATHOGEN INTERACTIONS TO CONTROL GRAFT-TRANSMISSIBLE DISEASES OF CITRUS

Sustainability of US agriculture depends on plant production and health. Plant pathogens cause significant economic losses in various crops. Among those isCitrus tristeza virus(CTV), the most severe viral pathogen of citrus.CTV causes two citrus diseases, which have had a major impact on global citrus production: quick decline (QD) or death of trees grown on the sour orange rootstock and stem pitting (SP). During the past century, epidemics of QD killed millions of trees on the sour orange rootstock in different citrus growing regions, including Florida. The SP disease caused by specific isolates of CTV severely affects tree growth and fruit quality of grapefruit and sweet orange, which are the two most economically important commodities for the citrus industry. The impact of SP on citrus production has been clearly documented in many different countries.At present, QD is managed with the use of resistant and/or tolerant rootstocks in combination with pathogen-free germplasm. However, sour orange is the best rootstock for fruit quality, is resistant to root rot diseases, and is adaptable to various soils. Thus, switching to the alternative rootstocks was not the ultimate solution for growers, and many Florida citrus producers are interested in bringing it back. SP is more difficult to control as it affects citrus varieties irrespective of the rootstock. The only means to protect commercial citrus varieties from SP is cross-protection with appropriate mild CTV isolates. This approach requires an understanding of the biology of CTV populations, and currently such understanding is insufficient.Despite the economic impact of CTV diseases, the mechanisms resulting in the manifestation of both diseases as well as the viral and host factors participating in these processes are still poorly understood, which hampers the development of means to manage the diseases in the field.There is also another important aspect for the need for better understanding the CTV pathogenicity. During the past decade, CTV has been developed into a transient expression vector, which is being prepared to be used in Florida for control of citrus greening (Huanglongbing, HLB). This devastating disease was first detected in Florida in 2005 and since thenhas spread throughout the citrus-growing regionsin many states in the US, including California.The vector carrying an anti-HLB gene can protect new plantings and can be inoculated into existing plantings to protect them against greening.It is possible that the vector can be used to treat trees already infected with HLB.Use of the CTV vector in commercial fields requires significant advancements in our knowledge of CTV pathogenicity as well as further improvement of vector effectiveness.The proposed study aims to advance our understanding of the CTV biology and provide a platform for the development of novel strategies to mitigate virus diseases and for the effective use of the CTV vector to control other citrus pathogens and pests.The research outcomes and findings of this project will be incorporated into a graduate-level course on Plant Virology in the Department of Plant Pathology, University of Florida. The proposed research activities willinvolve participation of new undergraduate and graduate students, including members of underrepresented groups.The research results will be broadly disseminated via publishing in the leading peer-reviewed journals and presentations at international, national, and local scientific meetings.

To accomplish the proposed objectives, we will use the advanced genomic, genetic, transcriptomic, and proteomic tools together with the innovative techniques for studying protein-protein interactions and ultrastructural approaches.Objective 1.We will conduct transcriptome analyses of host gene expression in CTV-infected citrus plants (an isolate of the T36 strain of CTV will be used) and evaluate which host genes are up- or down-regulated in response to the virus infection. The 12-15 months old seedlings of sweet orange Madam Vinouswill be inoculated with the infected budwood collected from the source trees infected with the T36 isolate, which have been maintained in our greenhouses. At three months post inoculation, plants will be tested by the enzyme-linked immunosorbent assay (ELISA) with the CTV-specific antibody to confirm the establishment of systemic infections (as per Sun and Folimonova, 2019). Tissue samples will be collected from newly growing flushes known to show most intensive virus replication and used to extract total RNA. The RNA preparations will be sent to Psomagen Inc. (Maryland, USA) for library preparation using the TruSeq Stranded mRNA Library prep (Illumina) Kit and sequencing on the NovaSeq 6000 S4 (Illumina) platform.We will engineerconstructs for ectopic expression of CTV proteins or protein domains, including the leader proteases, methyltransferase, helicase, and RNA-dependent RNA polymerase domains, p33, p6, p61, HSP70h, CPm, CP, p18, p13, p20, and p23. We will clone the genes of individual viral proteins in a binary vector pCASS4N and overexpress those using agroinfiltration-based transient expression or transgenic expression inNicotiana benthamianaand/or citrus. We will assess the phenotypes the proteins produce in those plants and which of these phenotypes correlate with the disease phenotypes induced by CTV. We will also conduct the examination of the interaction of the CTV proteins with the citrus host and of their role in disease production by using a yeast-two-hybrid cDNA library prepared using total RNA romCitrus macrophylla.Objective 2.To understand the rules and mechanisms of CTV cross-protection, we will examine viral factors that are involved in this phenomenon. We recently found that p33 binds a viral subgenomic RNA LMT1. We will characterize the specificity of this binding further and determine the domains of p33 as well as the structural features of LMT1 that determine their interaction using different biochemical approaches for the assessment of protein-RNA interactions. This will be followed by the examination of how disruption of p33-LMT1 binding affects cross-protection.Objective 3.This project objective is a collaborative effort with Dr. Hailing Jin, Professor at the University of California, Riverside. In this project, we will clone the nucleotide coding sequences of citrus-derived antimicrobial peptides (APs) provided by Dr. Jin into the insertion site of the CTV vector, which is located in the 3'-terminal region of the CTV genome and positioned under thenative subgenomic promoter of the CTV coat protein gene. The engineered constructs will be then inoculated into citrus trees and used as vehicles for delivery and expression of APs in citrus trees.The efficacy of APs against CLas will be assessed by challenge-inoculation of the AP-expressing citrus plants with the HLB-infected citrus tissue.