Publications

3841

Diaphorina citri serves as the primary vector for ‘ Candidatus Liberibacter asiaticus’ ( C Las), the bacterium associated with the severe Asian form of huanglongbing. C Las-positive D. citri are more fecund than their C Las-negative counterparts and require extra energy expenditure. Therefore, understanding the molecular mechanisms linking metabolism and reproduction is of particular importance. In this study, we found adipokinetic hormone ( DcAKH ) and its receptor ( DcAKHR ) were essential for increasing lipid metabolism and fecundity in response to C Las infection in D. citri. Knockdown of DcAKH and DcAKHR not only resulted in the accumulation of triacylglycerol and a decline of glycogen, but also significantly decreased fecundity and C Las titer in ovaries. Combined in vivo and in vitro experiments showed that miR-34 suppresses DcAKHR expression by binding to its 3’ untranslated region, whilst overexpression of miR-34 resulted in a decline of DcAKHR expression and C Las titer in ovaries and caused defects that mimicked DcAKHR knockdown phenotypes. Additionally, knockdown of DcAKH and DcAKHR significantly reduced juvenile hormone (JH) titer and JH signaling pathway genes in fat bodies and ovaries, including the JH receptor, methoprene-tolerant ( DcMet ), and the transcription factor, Krüppel homolog 1 (DcKr-h1) , that acts downstream of it, as well as the egg development related genes vitellogenin 1-like ( DcVg-1-like ), vitellogenin A1-like ( DcVg-A1-like ) and the vitellogenin receptor ( DcVgR ). As a result, C Las hijacks AKH/AKHR-miR-34-JH signaling to improve D. citri lipid metabolism and fecundity, while simultaneously increasing the replication of C Las, suggesting a mutualistic interaction between C Las and D. citri ovaries.

ABSTRACT The genome of “ Candidatus Xenohaliotis californiensis” was assembled from shotgun metagenomic sequencing of experimentally infected white abalone. Ninety-one percent genome completeness was achieved with low contamination. Sequencing this genome provides the opportunity to track pathogen evolution over time, conduct gene expression experiments, and study dynamics between this pathogen and its phage.

Strain LMG 33000T was isolated from a Bombus lapidarius gut sample. It shared the highest percentage 16S rRNA sequence identity, average amino acid identity, and amino acid identity of conserved genes with Convivina intestini LMG 28291T (95.86 %, 69.9 and 76.2 %, respectively), and the highest percentage OrthoANIu value with Fructobacillus fructosus DSM 20349T (71.4 %). Phylogenomic analyses by means of 107 or 120 conserved genes consistently revealed Convivina as nearest neighbour genus. The draft genome of strain LMG 33000T was 1.44 Mbp in size and had a DNA G+C content of 46.1 mol%. Genomic and physiological analyses revealed that strain LMG 33000T was a typical obligately fructophilic lactic acid bacterium that lacked the adhE and aldh genes and that did not produce ethanol during glucose or fructose metabolism. In contrast, Convivina species have the adhE and aldh genes in their genomes and produced ethanol from glucose and fructose metabolism, which is typical for heterofermentative lactic acid bacteria. Moreover, strain LMG 33000T exhibited catalase activity, an unusual characteristic among lactic acid bacteria, that is not shared with Convivina species. Given its position in the phylogenomic trees, and the difference in genomic percentage G+C content and in physiological and metabolic characteristics between strain LMG 33000T and Convivina species, we considered it most appropriate to classify strain LMG 33000T into a novel genus and species within the Lactobacillaceae family for which we propose the name Eupransor demetentiae gen. nov., sp. nov., with LMG 33000T (=CECT 30958T) as the type strain.

Citrus Huanglongbing, one of the most devastating citrus diseases, is caused by ‘ Candidatus Liberibacter asiaticus’ ( CLas). Polyamines are aliphatic nitrogen-containing compounds that play important roles in disease resistance and are synthesized primarily by two pathways: an arginine decarboxylation pathway and an ornithine decarboxylation pathway. However, it is unclear whether polyamines play a role in the tolerance of citrus to infection by CLas and, if so, whether one or both of the core polyamine metabolic pathways are important. We used high-performance liquid chromatography and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry to detect the contents of nine polyamine metabolism-related compounds in six citrus cultivars with varying levels of tolerance to CLas. We also systematically detected the changes in polyamine metabolism-related compounds and H2O2 contents and compared the gene expression levels and the activities of enzymes involved in the polyamine metabolic pathway among healthy, asymptomatic, and symptomatic leaves of Newhall navel oranges infected with CLas. The tolerant and moderately tolerant varieties showed higher polyamine metabolism-related compound levels than those of susceptible varieties. Compared with the healthy group, the symptomatic group showed significantly increased contents of arginine, ornithine, γ-aminobutyric acid, and putrescine by approximately 180, 19, 1.5, and 0.2 times, respectively, and upregulated expression of biosynthetic genes. Arginase and ornithine decarboxylase enzyme activities were the highest in the symptomatic group, whereas arginine decarboxylase and agmatine deiminase enzyme activities were the highest in the asymptomatic group. The two polyamine biosynthetic pathways showed different trends with the increase of the CLas titer, indicating that polyamines were mainly synthesized through the arginine decarboxylase pathway in the asymptomatic leaves and were synthesized via the ornithine decarboxylase pathway in symptomatic leaves. These findings provide new insight into the changes in polyamine metabolism in citrus infected with CLas.

Prophages/phages are important components of the genome of ‘Candidatus Liberibacter asiaticus’ (CLas), an unculturable alphaproteobacterium associated with citrus huanglongbing (HLB) disease. Phage variations have significant contributions to CLas strain diversity research, which provide critical information for HLB management. In this study, prophage variations among selected CLas strains from southern Texas were studied. The CLas strains were collected from three different CLas inhabitant environments: citrus leaf, citrus root, and Asian citrus psyllid (ACP), the vector of CLas. Regardless of the different habitats and time span, more than 80% of CLas strains consistently had both Type 1 and Type 2 prophages, the same prophage type profile as in CLas strains from Florida but different to those reported in California and China. Further studies were performed on prophage type diversity. Analyses on Type 1-specific PCR amplicon sequences (encoding an endolysin protein) revealed the presence of two groups: Type 1-A, clustered around prophage SC1 originating from Florida, and Type 1-B, clustered with prophage P-SGCA5-1 originating in California. Type 1-B strains were mostly from ACP of nearby citrus orchards. On the other hand, analyses on Type 2-specific PCR amplicon sequences (encoding a putative hypothetical protein) showed a single group clustering around prophage SC2 originated from Florida, although a different Type 2 prophage has been reported in California. The presence of two distinct Type 1 prophage groups suggested the possibility of two different CLas introductions in southern Texas. The results from this study provide an initial baseline of information on genomic and population diversity of CLas in Texas.