Contrasting amino acid profiles among permissive and non-permissive hosts of <i>Candidatus</i> Liberibacter asiaticus, putative causal agent of Huanglongbing

<div><p>Huanglongbing is a devastating disease of citrus. In this study, a comprehensive profile of phloem sap amino acids (AA) in four permissive host plants of <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las) and three non-permissive Rutaceae plants was conducted to gain a better understanding of host factors that may promote or suppress the bacterium. The AA profiles of <i>Diaphorina citri</i> nymphs and adults were similarly analyzed. A total of 38 unique AAs were detected in phloem sap of the various plants and <i>D</i>. <i>citri</i> samples, with phloem sap of young shoots containing more AAs and at higher concentrations than their mature counterparts. All AAs detected in phloem sap of non-permissive plants were also present in <i>C</i>Las -permissive hosts plus additional AAs in the latter class of plants. However, the relative composition of 18 commonly shared AAs varied between <i>C</i>Las -permissive hosts and non-permissive plants. Multivariate analysis with a partial least square discriminant methodology revealed a total of 12 AAs as major factors affecting <i>C</i>Las host status, of which seven were positively related to <i>C</i>Las tolerance/resistance and five positively associated with <i>C</i>Las susceptibility. Most of the AAs positively associated with <i>C</i>Las susceptibility were predominantly of the glutamate family, notably stressed-induced AAs such as arginine, GABA and proline. In contrast, AAs positively correlated with <i>C</i>Las tolerance/resistance were mainly of the serine family. Further analysis revealed that whereas the relative proportions of AAs positively associated with <i>C</i>Las susceptibility did not vary with host developmental stages, those associated with <i>C</i>Las tolerance/resistance increased with flush shoot maturity. Significantly, the proline-to-glycine ratio was determined to be an important discriminating factor for <i>C</i>Las permissivity with higher values characteristic of <i>C</i>Las -permissive hosts. This ratio could be exploited as a biomarker in HLB-resistance breeding programs.</p></div

Partial least squares-discriminant analysis (PLS-DA).

<p>Correlations between permissive and non-permissive hosts of <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las) and their free amino acid content as explanatory variables (X). The ellipse represents the Hotelling T² with 95% confidence interval (R²X_cum = 0.58, R²Y_cum = 0.99, Q²_cum = 0.79 for all hosts tested (A); R²X_cum = 0.64, R²Y_cum = 1, Q²_cum = 0.89 for <i>C</i>Las-permissive and non-permissive plants only (B). Q²_cum = cumulative fraction of the total variation of X’s that can be predicted by the extracted components; R²X_cum and R²Y_cum represent the fraction of the sum of squares of all X’s and Y’s explained by the current components, respectively. P = <i>C</i>Las-permissive and NP = <i>C</i>Las-non-permissive hosts defined as: OJY = young orange jasmine, OJM = mature orange jasmine, CLY = young curry leaf, CLM = mature curry leaf, WSY = young white sapote, WSM = mature white sapote, GFY = young grapefruit, GFM = mature grapefruit, SOY = young sweet orange, SOM = mature sweet orange, LEY = young lemon, LEM = mature lemon, PW = periwinkle plant, ACPAd = <i>D</i>. <i>citri</i> adults, and ACPNy = <i>D</i>. <i>citri</i> nymphs. The amino acids are defined as: AAAA, α-amino adipic acid; AABA, α-aminobutyric acid; Ala, alanine; Arg, arginine; Asn, asparagine; Asp, aspartate; BABA, β-aminobutyric acid; BALA, β-alanine; Carno, carnosine, Cit, citrulline; Cys, cystine; Cysta, cystathionine; Eth, ethanolamine; GABA, γ-aminobutyric acid; Gln, glutamine; Glu, glutamate; Gly, glycine; His, histidine; Hom, homocysteine; Hpro, hydroxyproline; Hydlys, hydroxyl-lysine; Ile, isoleucine; Leu, leucine; Lys, lysine; Meh, 1-methyl histidine; Met, methionine; Orn, ornithine; Phe, phenylalanine; PHOS, phosphoethanolamine; Orn, ornithine, Pro, proline; Sar, sarcosine; SEP, phosphoserine; Ser, serine; Tau, taurine; Thr, threonine; Trp, Tryptophan; Tyr, tyrosine; Val, valine.</p

Cluster heatmap describing the relative concentrations of free amino acids in phloem sap of permissive hosts of <i>Candidatus</i> Liberibacter asiaticus, non-permissive plants and whole <i>Diaphorina citri</i> nymphs and adults (OJY = young orange jasmine, OJM = mature orange jasmine, CLY = young curry leaf, CLM = mature curry leaf, WSY = young white sapote, WSM = mature white sapote, GFY = young grapefruit, GFM = mature grapefruit, SOY = young sweet orange, SOM = mature sweet orange, LEY = young lemon, LEM = mature lemon, PW = periwinkle plant, ACPAd = <i>D</i>. <i>citri</i> adults, and ACPNy = <i>D</i>. <i>citri</i> nymphs).

<p>Cluster heatmap describing the relative concentrations of free amino acids in phloem sap of permissive hosts of <i>Candidatus</i> Liberibacter asiaticus, non-permissive plants and whole <i>Diaphorina citri</i> nymphs and adults (OJY = young orange jasmine, OJM = mature orange jasmine, CLY = young curry leaf, CLM = mature curry leaf, WSY = young white sapote, WSM = mature white sapote, GFY = young grapefruit, GFM = mature grapefruit, SOY = young sweet orange, SOM = mature sweet orange, LEY = young lemon, LEM = mature lemon, PW = periwinkle plant, ACPAd = <i>D</i>. <i>citri</i> adults, and ACPNy = <i>D</i>. <i>citri</i> nymphs).</p

Correlation between mean amino acid concentrations and <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las) permissivity status of different plants and <i>Diaphorina citri</i> (ACP) adults and nymphs.

<p>Correlation between mean amino acid concentrations and <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las) permissivity status of different plants and <i>Diaphorina citri</i> (ACP) adults and nymphs.</p

Venn diagram comparing shared and unique free amino acids present in phloem sap of non-permissive (curry leaf, orange jasmine and white sapote), and permissive (grapefruit, lemon and sweet orange) hosts of <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las).

<p>A = young shoots and B = mature shoots.</p

Relative concentrations of free amino acids detected in phloem sap of young and mature Rutaceae flush shoots, periwinkle plants, and whole <i>Diaphorina citri</i> nymphs and adults populations.

<p>Relative concentrations of free amino acids detected in phloem sap of young and mature Rutaceae flush shoots, periwinkle plants, and whole <i>Diaphorina citri</i> nymphs and adults populations.</p

List of plant species evaluated in this study and their permissiveness to <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las) or the Asian citrus psyllid (ACP).

<p>List of plant species evaluated in this study and their permissiveness to <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las) or the Asian citrus psyllid (ACP).</p

Ratio of mean concentrations between <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las)-permissive and non–permissive hosts for key amino acids identified as variables of most importance in partial least squares-discriminant analysis (PLS-DA).

<p>Ratio of mean concentrations between <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las)-permissive and non–permissive hosts for key amino acids identified as variables of most importance in partial least squares-discriminant analysis (PLS-DA).</p

Mean amino acid concentration¹(μg/g) of leaf tissue of <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las)-permissive and non–permissive plants² and of whole <i>Diaphorina citri</i> (ACP) adults and nymphs analyzed by HPLC.

<p>Mean amino acid concentration<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187921#t002fn001" target="_blank">¹</a>(μg/g) of leaf tissue of <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las)-permissive and non–permissive plants<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187921#t002fn002" target="_blank">²</a> and of whole <i>Diaphorina citri</i> (ACP) adults and nymphs analyzed by HPLC.</p

Free amino acid profiles of permissive hosts (P) of <i>Candidatus</i> Liberibacter asiaticus (<i>C</i>Las) and non-permissive (NP) plants.

<p>Permissive hosts comprise phloem sap of young and mature flush shoots of three citrus species, periwinkle plants and whole <i>Diaphorina citri</i> nymphs and adults, while non-permissive host comprise young and mature shoots of curry leaf, orange jasmine and white sapote. Results are based on mean values obtained for growth stages of each plant species (three replicates) and are shown as the ratios of accumulation between the two host categories. Asterisk indicates significant differences according to t-test (P<0.05).</p