Dissecting The Function Of Susceptibility Gene CsLOB1 Of Citrus Bacterial Canker Disease

Citrus canker,caused by Xanthomonas citri ssp.citri?Xcc?,is a devastating disease worldwide that infects most commercial citrus varieties through stomata and wounds.The typical necrotic lesions occur on the fruits,leaves,and stem,which affect citrus quality and yield.PthA4,a transcription activator-like?TAL?effector translocated by the type III secretion system?T3SS?,is a critical pathogenicity determinant of Xanthomonas citri subsp.citri strain Xcc306.Previous research showed that knocking out of pthA4 results in loss of pathogenicity in sweet orange and grapefruit.PthA4 induces the expression of specific disease susceptibility genes during infection by binding to Effector-Binding-Elements?EBEs?within the promoter regions of the susceptibility genes.Expression of genes involved in cell wall modification,DNA packaging,and cell division were significantly up-regulated by PthA4.By using Microarray analysis and PthA4 target prediction,it has been suggested that PthA4targets CsLOB1,a plant-specific transcriptional factor in the lateral organ boundaries?LOB?domain family,to induce its expression and downstream genes to cause pustule formation.Meanwhile,designed TALEs targeting CsLOB1 complemented citrus canker symptoms caused by Xcc306?pthA4 in sweet orange.Experimental and transcriptional data support that CsLOB1 is the putative host target gene of PthA4.We expected to speculate the gene function of CsLOB1 in the molecular pathogenicity mechanism of Xcc.In this dissertation,I have reported the following results:Confirmation of gene expression induced by PthA4.To confirm microarray data?sweet orange infected by Xcc306::sweet orange infected by Xcc306?pthA4?,we detected the expression of PthA4-induced genes by using a quantitative reverse transcription-PCR?qRT-PCR?.The result showed a similar gene expression trend between microarray and qRT-PCR results.Linear regression analysis reveals the coefficient value to be 0.89,supporting the reliability of the microarray data for further research.Protein analysis of Citrus LBD family.To explore the molecular characteristics of LBD family,we identified 36 LOB domain containing?LBD?proteins in the HuaZhong Citrus Sinensis database.We found that CsLOB1 contains a conserved C block which contains four Cysteine residues in a CX₂CX₆CX₃C zinc finger-like motif,an invariant Glycine residue harboring a Glycine-Alanine-Serine Block?GAS?block and coiled-coil structures harboring an LX₆LX₃LX₆L zipper-like motif at C-terminus.Tissue expression pattern of the CsLOB1 gene.To investigate the expression pattern of CsLOB1 in citrus,we tested its expression level in flower,root,stem and leaves of Valencia sweet orange by using qRT-PCR analysis.The expression level of CsLOB1 was higher in leaves and stems than in flowers and roots.Subcellular localization analyses of CsLOB1.To test the subcellular localization of CsLOB1 in citrus cells,we transiently expressed 35S:EYFP-CsLOB1in citrus protoplast.Fluorescence microscopy analyses of GFP and DAPI indicate that CsLOB1 was primarily localized in the nucleus.Furthermore,transient expression of mCherry-NLS,which localizes to the nucleus,overlapped with 35S:EYFP-CsLOB1when co-transformed into citrus protoplast,further supporting CsLOB1 localizes in the nucleus.CsLOB1 effects plant growth and development in citrus.Citrus tristeza virus-induced gene silencing?VIGS?plants were constructed to suppress the gene expression of CsLOB1 in Alemow?Citrus macrophylla Wester?.The growth and development of VIGS plants were slower compared to negative control plants.Reduced citrus canker resistance was observed on VIGS leaves inoculated with Xcc306 at the concentration of 5x10⁴cfu/mL.The unusual microstructure of VIGS plants were captured under different kinds of microscopes,but not in negative control plants.Ectopic CsLOB1 expression in citrus induces pustule lesion.To establish a direct causal relationship between citrus canker symptoms and expression of CsLOB1,we constructed the ectopic CsLOB1 overexpression transgenic Duncan grapefruit?Citrus paradisi Macf.?,which takes advantage of the synthetic glucocorticoid-induced nuclear targeting of a reporter construct containing glucocorticoid receptor?GR?.Dexthemasone?DEX?treated CsLOB1-GR-transgenic plants exhibited a pustule formation on the leaves.,however,the pustule symptoms were not observed in DEX-treated negative control plants and non-treated CsLOB1-transgenic plants.Ectopic CsLOB1 expression in citrus restored water soaking symptom by Xcc306?pthA4.To confirm that CsLOB1 is the susceptibility gene of Xcc in citrus,transgenic Duncan grapefruit?Citrus paradisi Macf.?plants expressing 35S:CsLOB1-GR were generated to test the symptoms on the leaves inoculated with Xcc306?pthA4.When CsLOB1-transgenic Duncan grapefruit leaves were inoculated with Xcc306?pthA4,then treated with DEX solution and mock at 24 hpi,water-soaking symptoms were observed on DEX-treated transgenic plant leaves,but not on mock treated plants at seven days post inoculation.Interestingly,slightly water soaking was observed with those pustule symptoms in DEX-treated CsLOB1-transgenic plants.CsLOB1 induces PthA4-iduced genes.To test the hypothesis that ectopic expression of CsLOB1 will induce PthA4-induced genes,q RT-PCR was applied to detect the gene expression of PthA4-induced genes in DEX-treated transgenic plants.The result showed CsLOB1 induced 12 genes of those PthA4-induced genes.