The Destructive Citrus Pathogen, ‘<em>Candidatus</em> Liberibacter asiaticus’ Encodes a Functional Flagellin Characteristic of a Pathogen-Associated Molecular Pattern

<div><p>Huanglongbing (HLB) is presently the most devastating citrus disease worldwide. As an intracellular plant pathogen and insect symbiont, the HLB bacterium, ‘<em>Candidatus</em> Liberibacter asiaticus’ (Las), retains the entire flagellum-encoding gene cluster in its significantly reduced genome. Las encodes a flagellin and hook-associated protein (Fla) of 452 amino acids that contains a conserved 22 amino acid domain (flg22) at positions 29 to 50 in the N-terminus. The phenotypic alteration in motility of a <em>Sinorhizobium meliloti</em> mutant lacking the <em>fla</em> genes was partially restored by constitutive expression of Fla<em>_Las</em>. <em>Agrobacterium</em>-mediated transient expression <em>in planta</em> revealed that Fla<em>_Las</em> induced cell death and callose deposition in <em>Nicotiana benthamiana</em>, and that the transcription of <em>BAK1</em> and <em>SGT1</em>, which are associated with plant innate immunity, was upregulated. Amino acid substitution experiments revealed that residues 38 (serine) and 39 (aspartate) of Fla<em>_Las</em> were essential for callose induction. The synthetic flg22<em>_Las</em> peptide could not induce plant cell death but retained the ability to induce callose deposition at a concentration of 20 µM or above. This demonstrated that the pathogen-associated molecular pattern (PAMP) activity of flg22 in Las was weaker than those in other well-studied plant pathogenic bacteria. These results indicate that Fla<em>_Las</em> acts as a PAMP and may play an important role in triggering host plant resistance to the HLB bacteria.</p> </div

Table_2_The ColRS-Regulated Membrane Protein Gene XAC1347 Is Involved in Copper Homeostasis and hrp Gene Expression in Xanthomonas citri subsp. citri.DOCX

<p>Xanthomonas citri subsp. citri (Xcc) is the major causal agent of citrus canker disease. The XAC1347 gene, which encodes a conserved membrane protein in Xcc, is required for virulence during infection. However, the molecular events mediated by XAC1347 remain unclear. In this study, we reported that XAC1347 gene is positively regulated by two component regulatory system ColRS and required for type III secretion system function. A non-polar deletion mutant of the XAC1347 gene resulted in a Hrp minus phenotype in plants and reduced copper homeostasis. Real-time PCR experiments indicated that XAC1347 gene is induced by copper ions. The expression levels of representative genes from four hrp operons, including hrpB1, hrcV, hrpF, and hrpD6, were reduced in XAC1347 mutant, indicating that XAC1347 is involved hrp gene expression.</p

Bacterial growth in citrus tissue.

<p>Leaf planchets were cut from each infiltration area at 2-d intervals post inoculation to analyze bacterial growth. The values shown are means of three technical repeats with standard deviations.</p

Expression of cellulase genes in <i>X</i>. <i>citri</i> subsp. <i>citri</i>.

<p>(A) RT-PCR analysis of gene transcription in <i>X</i>. <i>citri</i> subsp. <i>citri</i> culturing in nutrient broth medium (NB) and growing in citrus. M, DL2000 marker. (B) Detection of BglC3 and EngXCA secretion using immunoblotting. Protein samples were analyzed by SDS-PAGE and immunoblotted using anti-c-Myc antibodies. TE, Total protein extract; SE, Culture supernatant.</p

Image_4_The ColRS-Regulated Membrane Protein Gene XAC1347 Is Involved in Copper Homeostasis and hrp Gene Expression in Xanthomonas citri subsp. citri.TIF

<p>Xanthomonas citri subsp. citri (Xcc) is the major causal agent of citrus canker disease. The XAC1347 gene, which encodes a conserved membrane protein in Xcc, is required for virulence during infection. However, the molecular events mediated by XAC1347 remain unclear. In this study, we reported that XAC1347 gene is positively regulated by two component regulatory system ColRS and required for type III secretion system function. A non-polar deletion mutant of the XAC1347 gene resulted in a Hrp minus phenotype in plants and reduced copper homeostasis. Real-time PCR experiments indicated that XAC1347 gene is induced by copper ions. The expression levels of representative genes from four hrp operons, including hrpB1, hrcV, hrpF, and hrpD6, were reduced in XAC1347 mutant, indicating that XAC1347 is involved hrp gene expression.</p

Image_2_The ColRS-Regulated Membrane Protein Gene XAC1347 Is Involved in Copper Homeostasis and hrp Gene Expression in Xanthomonas citri subsp. citri.TIF

<p>Xanthomonas citri subsp. citri (Xcc) is the major causal agent of citrus canker disease. The XAC1347 gene, which encodes a conserved membrane protein in Xcc, is required for virulence during infection. However, the molecular events mediated by XAC1347 remain unclear. In this study, we reported that XAC1347 gene is positively regulated by two component regulatory system ColRS and required for type III secretion system function. A non-polar deletion mutant of the XAC1347 gene resulted in a Hrp minus phenotype in plants and reduced copper homeostasis. Real-time PCR experiments indicated that XAC1347 gene is induced by copper ions. The expression levels of representative genes from four hrp operons, including hrpB1, hrcV, hrpF, and hrpD6, were reduced in XAC1347 mutant, indicating that XAC1347 is involved hrp gene expression.</p

Image_3_The ColRS-Regulated Membrane Protein Gene XAC1347 Is Involved in Copper Homeostasis and hrp Gene Expression in Xanthomonas citri subsp. citri.TIF

<p>Xanthomonas citri subsp. citri (Xcc) is the major causal agent of citrus canker disease. The XAC1347 gene, which encodes a conserved membrane protein in Xcc, is required for virulence during infection. However, the molecular events mediated by XAC1347 remain unclear. In this study, we reported that XAC1347 gene is positively regulated by two component regulatory system ColRS and required for type III secretion system function. A non-polar deletion mutant of the XAC1347 gene resulted in a Hrp minus phenotype in plants and reduced copper homeostasis. Real-time PCR experiments indicated that XAC1347 gene is induced by copper ions. The expression levels of representative genes from four hrp operons, including hrpB1, hrcV, hrpF, and hrpD6, were reduced in XAC1347 mutant, indicating that XAC1347 is involved hrp gene expression.</p

Table_3_The ColRS-Regulated Membrane Protein Gene XAC1347 Is Involved in Copper Homeostasis and hrp Gene Expression in Xanthomonas citri subsp. citri.DOCX

<p>Xanthomonas citri subsp. citri (Xcc) is the major causal agent of citrus canker disease. The XAC1347 gene, which encodes a conserved membrane protein in Xcc, is required for virulence during infection. However, the molecular events mediated by XAC1347 remain unclear. In this study, we reported that XAC1347 gene is positively regulated by two component regulatory system ColRS and required for type III secretion system function. A non-polar deletion mutant of the XAC1347 gene resulted in a Hrp minus phenotype in plants and reduced copper homeostasis. Real-time PCR experiments indicated that XAC1347 gene is induced by copper ions. The expression levels of representative genes from four hrp operons, including hrpB1, hrcV, hrpF, and hrpD6, were reduced in XAC1347 mutant, indicating that XAC1347 is involved hrp gene expression.</p

Minimum concentration of flg22 <i>_Las</i> need for callose deposition.

<p>Synthetic peptide was diluted with double distilled water to a final concentrations of 5, 10, 15, 20, 25, 30 35 and 40 µM and infiltrated into tobacco leaves. Callose deposition was examined at 10 days later. The experiments were performed in three independent replicates.</p

Impact of amino acid changes at positions R30, S38 and D39 in <i>Ca.</i> Liberibacter asiaticus (Las) flagellin’s conserved domain, flg22<i>_Las</i>, on callose deposition. A,

<p>Amino acid changes made in flg22<i>_Las</i>. The 30^th, 38^th and 39^th amino acid residues were changed to histidine, glycine and glutamine by a primer extension strategy. <b>B,</b> Callose deposition. PCR products were inserted into the potato virus X vector pgR107 for Agro-infiltration. Callose deposition was visualized under UV epifluorescence at 12 hours after infiltration. The results shown are representative of data from four independent replicates.</p