CHARACTERIZE THE VIRULENCE MECHANISM OF CITRUS HLB PATHOGEN CANDIDATUS LIBERIBACTER ASIATICUS AND MANAGEMENT OF HLB BY GENERATING HLB RESISTANT/TOLERANT CITRUS VARIETIES

The goal is to understand how citrus defends itself against Candidatus Liberibacter asiaticus (Las) infection and how Las overcomes the plant defense. It has been observed that Las causes delayed plant defense response which eventually leads to phloem necrosis. Citrus defense response has been suggested to result from recognition of pathogen-associated molecular patterns (PAMPs) or effectors of Las. The delayed plant defense response probably results from the effect of some effectors. Here, we aim to identify the receptors of PAMPs (e.g., flagellin, pili, outer membrane proteins, peptidoglycan, elongation factor Tu) in Valencia sweet orange (HLB susceptible) and in HLB tolerant trifoliate orange (Poncirus trifoliata). We will overexpress the receptors in Valencia sweet orange using 35S promoter. We will conduct cisgenic expression of Poncirus receptors in susceptible citrus varieties. We will determine whether the transgenic or cisgenic citrus plants have increased resistance against Las. We will further investigate the putative effectors of Las regarding their contribution to disease development and suppressing plant defense. We will use Liberibacter crescens as a model to confirm their secretion. For the confirmed effectors, we will conduct Y2H to identify their targets in sweet orange and Poncirus. We will conduct genome editing of putative susceptible genes to HLB and test whether the genome modified plants have increased HLB resistance. The proposed study will contribute to long-term solution of HLB control via developing HLB resistant citrus varieties.

Objective 1. 1.1. Y2H assayIn our previous study, we have conducted a proof-of-concept test to identify PAMP receptors. One of the outer membrane proteins is shown to interact with orange 1.1t04683 that encodes a TIR-NBS-LRR resistance protein muRdr1C. TIR-NBS-LRR proteins might function as receptors that bind to molecules secreted by pathogens (McHale et al., 2006). We will continue to identify the targets or receptors for other outer membrane proteins, pili, flagellin, Elongation factor Tu, and peptidoglycan using both Y2H and surface plasmon resonance methods. Y2H will be conducted following the manufacturer's instructions and as detailed above.1.2. Surface plasmon resonance (SPR) assayFor the SPR assay, we will conduct the analysis as described previously (Madeira et al., 2009; Madeira et al., 2011).1.3. Confirmation using GST pull-down assay and bimolecular fluorescence complementation (BiFC) assay to corroborate any interactions?Objective 2.For the PAMP receptors in HLB susceptible Valencia sweet orange and tolerant Poncirus. identified in Objective 1, we will conduct the following:If the receptors for the same PAMPs are different in Valencia and Poncirus, we will clone the receptors from Poncirus and express them in Valencia via Agrobacterium-mediated transformation as reported previously (Orbovi? and Grosser, 2015) using 35S promoter, phloem specific promoters (e.g., AtSuc2) or native promoters from Poncirus and test whether the overexpression increases plant defense to Las. We will further confirm the cisgenic plants by qPCR and Western blot.If the receptors for the same PAMPs are the same in Valencia sweet orange and Poncirus, we will compare the expression of receptor genes to Las infection. For receptor genes with differential expression in Valencia and Poncirus, we will compare the promoter sequence. If the promoter sequence is different, we will replace the promoter sequences of the corresponding receptor genes in Valencia with that in Poncirus using the CRISPR/Cas mediated genome editing.The HLB resistance of the transgenic/cisgenic plants will be examined after inoculation of Las via grafting or psyllid transmission and will be compared to wild-type plant. Las titer and disease severity in genome-modified and control citrus plants will be evaluated at 6, 12, 18, and 24 months after infection with Las as described previously (Li et al., 2015).Objective 3.3.1. Confirmation of the secretion of Sec-dependent effectorsIn this objective, we will use Sinorhizobium meliloti or Agrobacterium tumefaciens as a model because Las has not been cultured in media. pUFR053 is able to multiply in Sinorhizobium meliloti or Agrobacterium tumefaciens. We will express putative Las effectors with FLAG, HA or GST tags using pUFR053 in Sinorhizobium meliloti or Agrobacterium tumefaciens and detect their secretion into the medium. Briefly, the full sequence of SDE genes will be amplified and cloned in the pUFR053 vector. For the isolation of secreted protein from culture supernatant, Sinorhizobium meliloti or Agrobacterium tumefaciens cultures will be grown in 100 ml of BM7 liquid medium until OD600 reached 0.3-0.4. Bacterial cells will be removed by centrifugation (5 min at 4,000 × g) and filtration of the supernatant through a 0.45-μm-pore-size filter. Protein from cell-free culture supernatants will be precipitated by addition of trichloroacetic acid (10% [wt/vol] final concentration), recovered by centrifugation for 1 h at 10,000 × g, and washed twice with acetone. Identification of the secreted proteins will be confirmed using western blot with appropriate antibodies against FLAG, HA or GST tags.3.2. Determining whether the identified effectors are involved in suppressing plant defenseTo determine whether the identified effectors are involved in suppressing plant defense, we will employ the following assays. First, we will test whether SDEs suppress plant defense responses induced by effectors. Briefly, we will infiltrate the leaves of Nicotiana benthamiana with Agrobacterium harboring the binary vector containing SDE-EYFP fusion protein under the 35S promoter, followed by infiltration with Agrobacterium containing an Xanthomonas effector AvrBsT at 2 days after SDE infiltration. AvrBsT is known to induce HR in N. benthamiana (Kim et al., 2010). Second, we will test whether the effectors suppress PTI. For this assay, we will infiltrate the leaves transiently expressing SDEs with A. tumefaciens GV3101 with FlaLas. Transient expression of FlaLas in N. benthamiana is known to induce plant defense responses (Hao et al., 2014; Shi et al., 2018). Similarly, we will test the suppressing effect of effectors on other PAMPs.3.3. Identification of SDE targets in PoncirusFor the putative Sec-dependent effectors (SDEs) (Prasad et al., 2016), we will conduct Y2H and SPR assays to identify their targets in Valencia and Poncirus. The GST pull-down assay and BiFC assay will be conducted to corroborate any interactions.3.4. Genetic modification of ValenciaWe will compare the SDE targets in Valencia and Poncirus. If the targets are different for the same SDEs in Valencia and Poncirus, we will express the correspondent Poncirus genes in Valencia and test the transgenic/cisgenic plants against Las as described in Objective 2. If necessary, we can mutate the native target genes in Valencia as described previously (Jia et al., 2017). We will compare the resistance of wild type Valencia, Valencia expressing Poncirus targets, Valencia mutant in native targets, and Valencia mutant expressing Poncirus target gene against HLB and evaluate other horticultural traits as described in Objective 2.If the targets are the same for the same SDEs in Valencia and Poncirus, we will compare their gene expression to Las infection. For target genes with different expression in Valencia and Poncirus, we will compare their promoter regions. If the promoter region is different, we will replace the corresponding promoter region of Valencia with that of Poncirus using CRISPR/Cas9 technology with the promoter region as a donor. We will test the resistance of the genome modified plants against HLB and evaluate other horticultural traits as described in Objective 2.