Effector enrichment by Candidatus Liberibacter promotes Diaphorina citri feeding via Jasmonic acid pathway suppression.

BACKGROUND: Citrus huanglongbing (HLB) is a devastating disease caused by Candidatus Liberibacter asiaticus (CLas) that affects the citrus industry. In nature, CLas relies primarily on Diaphorina citri Kuwayama as its vector for dissemination. After D. citri ingests CLas-infected citrus, the pathogen infiltrates the insect's body, where it thrives, reproduces, and exerts regulatory control over the growth and metabolism of D. citri. Previous studies have shown that CLas alters the composition of proteins in the saliva of D. citri, but the functions of these proteins remain largely unknown. RESULTS: In this study, we detected two proteins (DcitSGP1 and DcitSGP3) with high expression levels in CLas-infected D. citri. Quantitative PCR and Western blotting analysis showed that the two proteins were highly expressed in the salivary glands and delivered into the host plant during feeding. Silencing the two genes significantly decreased the survival rate for D. citri, reduced phloem nutrition sucking and promoted jasmonic acid (JA) defenses in citrus. By contrast, after overexpressing the two genes in citrus, the expression levels of JA pathway-associated genes decreased. CONCLUSION: Our results suggest that CLas can indirectly suppress the defenses of citrus and support feeding by D. citri via increasing the levels of effectors in the insect's saliva. This discovery facilitates further research into the interaction between insect vectors and pathogens. © 2024 Society of Chemical Industry.

Regulation of soldier caste differentiation by microRNAs in Formosan subterranean termite (Coptotermes formosanus Shiraki).

The soldier caste is one of the most distinguished castes inside the termite colony. The mechanism of soldier caste differentiation has mainly been studied at the transcriptional level, but the function of microRNAs (miRNAs) in soldier caste differentiation is seldom studied. In this study, the workers of Coptotermes formosanus Shiraki were treated with methoprene, a juvenile hormone analog which can induce workers to transform into soldiers. The miRNomes of the methoprene-treated workers and the controls were sequenced. Then, the differentially expressed miRNAs (DEmiRs) were corrected with the differentially expressed genes DEGs to construct the DEmiR-DEG regulatory network. Afterwards, the DEmiR-regulated DEGs were subjected to GO enrichment and KEGG enrichment analysis. A total of 1,324 miRNAs were identified, among which 116 miRNAs were screened as DEmiRs between the methoprene-treated group and the control group. A total of 4,433 DEmiR-DEG pairs were obtained. No GO term was recognized as significant in the cellular component, molecular function, or biological process categories. The KEGG enrichment analysis of the DEmiR-regulated DEGs showed that the ribosome biogenesis in eukaryotes and circadian rhythm-fly pathways were enriched. This study demonstrates that DEmiRs and DEGs form a complex network regulating soldier caste differentiation in termites.

Identification of the pheromone biosynthesis genes from the sex pheromone gland transcriptome of the diamondback moth, Plutella xylostella.

The diamondback moth was estimated to increase costs to the global agricultural economy as the global area increase of Brassica vegetable crops and oilseed rape. Sex pheromones traps are outstanding tools available in Integrated Pest Management for many years and provides an effective approach for DBM population monitoring and control. The ratio of two major sex pheromone compounds shows geographical variations. However, the limitation of our information in the DBM pheromone biosynthesis dampens our understanding of the ratio diversity of pheromone compounds. Here, we constructed a transcriptomic library from the DBM pheromone gland and identified genes putatively involved in the fatty acid biosynthesis, pheromones functional group transfer, and ß-oxidation enzymes. In addition, odorant binding protein, chemosensory protein and pheromone binding protein genes encoded in the pheromone gland transcriptome, suggest that female DBM moths may receive odors or pheromone compounds via their pheromone gland and ovipositor system. Tissue expression profiles further revealed that two ALR, three DES and one FAR5 genes were pheromone gland tissue biased, while some chemoreception genes expressed extensively in PG, pupa, antenna and legs tissues. Finally, the candidate genes from large-scale transcriptome information may be useful for characterizing a presumed biosynthetic pathway of the DBM sex pheromone.

Molecular identification of differential expression genes associated with sex pheromone biosynthesis in Spodoptera exigua.

Species-specific sex pheromone is biosynthesized and released in most female moths as a chemical cue in mating communication. However, information on genes involved in this pathway is limited. The beet armyworm, Spodoptera exigua, is a cosmopolitan agricultural pest that causes severe economic losses to many crops. In China, the female sex pheromones in sex pheromone glands (PGs) of S. exigua have been measured which comprises (Z,E)-9,12-tetradecadienyl acetate, (Z)-9-tetradecen-l-ol, (Z)-9-tetradecenyl acetate, and (Z,E)-9,12-tetradecadien-1-ol in a ratio of 47:18:18:17. Fifty-nine putative genes related to sex pheromone biosynthesis were identified in the present study by sequencing and analyzing the sex pheromone gland (PG) transcriptome of S. exigua. Expression profiles revealed that two desaturase (SexiDes5 and SexiDes11) and three fatty acyl reductase (SexiFAR2, 3, and 9) genes had PG-specific expression, and phylogenetic analysis demonstrated that they clustered with genes known to be involved in pheromone synthesis in other moth species. Our results provide crucial background information that could facilitate the elucidation of sex pheromone biosynthesis pathway of S. exigua as well as other Spodoptera species and help identify potential targets for disrupting sexual communication in S. exigua for developing novel environment-friendly pesticides.

The complete mitochondrial genome of Tetranychus truncatus Ehara (Acari: Tetranychidae).

The complete mitochondrial genome of Tetranychus truncatus Ehara (Acari: Tetranychidae) is a typical circular DNA with length of 13,089 bp (GenBank accession number: KM111296). The genome contains all 13 protein-coding genes (PCGs), an A + T-rich region, two rRNA genes and 22 tRNA genes. The A + T content of the mitochondrial genome is 84.5%. The AT-skew is positive (0.032) while the GC-skew is negative (-0.058). The gene arrangement is conserved in T. urticae, Panonychus citri and P. ulmi which are in the same family (Tetranychidae). The A + T-rich region is only 43 bp in length with high A + T content (97.7%). All the PCGs start with typical ATD codons. Eight PCGs have complete TAA stop codons, while five PCGs have an incomplete stop codon (T).

Infection Rate Assay by Nested PCR and the Phylogenetic Analysis of Himetobi P Virus in the Main Pests of Rice-Wheat Cropping Systems.

Himetobi P virus (HiPV) is an ssRNA in the family Dicistroviridae that infects rice pests belonging to Hemiptera. To determine its host range, a nested PCR method was designed to detect HiPV in some of the main rice pests (Hemiptera) in eastern China. The incidence of infection in the grain aphid Sitobion avenae Fabricius (Hemiptera: Aphididae) was low (3%), while high incidences of infection occurred in the planthoppers Laodelphax striatellus (Fallén) (Hemiptera: Delphacidae) (100%) and Nilaparvata lugens (Hemiptera: Delphacidae) (51%) and in the leafhoppers Cicadella viridis (Hemiptera: Cicadellidae) (90%) and Nephotettix cincticeps (Hemiptera: Cicadellidae) (57%). Phylogenetic analysis by maximum likelihood tree and median-joining networks implied the HiPVs from the same hosts were genetically close. Neutral equilibrium evolution for the polymorphism data was tested by the Tajima's D test and by Fu and Li's D and F tests. Test values were negative, which indicates a selection on the HiPV haplotypes. We sequenced the complete genome sequence of HiPV to look for evidence of recombination. We identified a recombination event in which two genomes recombined in the region of ORF2. The two open reading frames of the HiPV had been selected with low Ka/Ks ratios compared with two previous genome sequences.

Identification of a core set of rhizobial infection genes using data from single cell-types.

Genome-wide expression studies on nodulation have varied in their scale from entire root systems to dissected nodules or root sections containing nodule primordia (NP). More recently efforts have focused on developing methods for isolation of root hairs from infected plants and the application of laser-capture microdissection technology to nodules. Here we analyze two published data sets to identify a core set of infection genes that are expressed in the nodule and in root hairs during infection. Among the genes identified were those encoding phenylpropanoid biosynthesis enzymes including Chalcone-O-Methyltransferase which is required for the production of the potent Nod gene inducer 4',4-dihydroxy-2-methoxychalcone. A promoter-GUS analysis in transgenic hairy roots for two genes encoding Chalcone-O-Methyltransferase isoforms revealed their expression in rhizobially infected root hairs and the nodule infection zone but not in the nitrogen fixation zone. We also describe a group of Rhizobially Induced Peroxidases whose expression overlaps with the production of superoxide in rhizobially infected root hairs and in nodules and roots. Finally, we identify a cohort of co-regulated transcription factors as candidate regulators of these processes.

The complete mitochondrial genomes of six species of Tetranychus provide insights into the phylogeny and evolution of spider mites.

Many spider mites belonging to the genus Tetranychus are of agronomical importance. With limited morphological characters, Tetranychus mites are usually identified by a combination of morphological characteristics and molecular diagnostics. To clarify their molecular evolution and phylogeny, the mitochondrial genomes of the green and red forms of Tetranychus urticae as well as T. kanzawai, T. ludeni, T. malaysiensis, T. phaselus, T. pueraricola were sequenced and compared. The seven mitochondrial genomes are typical circular molecules of about 13,000 bp encoding and they are composed of the complete set of 37 genes that are usually found in metazoans. The order of the mitochondrial (mt) genes is the same as that in the mt genomes of Panonychus citri and P. ulmi, but very different from that in other Acari. The J-strands of the mitochondrial genomes have high (∼ 84%) A+T contents, negative GC-skews and positive AT-skews. The nucleotide sequence of the cox1 gene, which is commonly used as a taxon barcode and molecular marker, is more highly conserved than the nucleotide sequences of other mitochondrial genes in these seven species. Most tRNA genes in the seven genomes lose the D-arm and/or the T-arm. The functions of these tRNAs need to be evaluated. The mitochondrial genome of T. malaysiensis differs from the other six genomes in having a slightly smaller genome size, a slight difference in codon usage, and a variable loop in place of the T-arm of some tRNAs by a variable loop. A phylogenic analysis shows that T. malaysiensis first split from other Tetranychus species and that the clade of the family Tetranychoidea occupies a basal position in the Trombidiformes. The mt genomes of the green and red forms of T. urticae have limited divergence and short evolutionary distance.

Wolbachia-Host Interactions: Host Mating Patterns Affect Wolbachia Density Dynamics.

Wolbachia are maternally inherited intracellular bacteria that infect a wide range of arthropods and cause an array of effects on host reproduction, fitness and mating behavior. Although our understanding of the Wolbachia-associated effects on hosts is rapidly expanding, our knowledge of the host factors that mediate Wolbachia dynamics is rudimentary. Here, we explore the interactions between Wolbachia and its host, the two-spotted spider mite Tetranychus urticae Koch. Our results indicate that Wolbachia induces strong cytoplasmic incompatibility (CI), increases host fecundity, but has no effects on the longevity of females and the mating competitiveness of males in T. urticae. Most importantly, host mating pattern was found to affect Wolbachia density dynamics during host aging. Mating of an uninfected mite of either sex with an infected mite attenuates the Wolbachia density in the infected mite. According to the results of Wolbachia localization, this finding may be associated with the tropism of Wolbachia for the reproductive tissue in adult spider mites. Our findings describe a new interaction between Wolbachia and their hosts.

The complete mitochondrial genomes of two rice planthoppers, Nilaparvata lugens and Laodelphax striatellus: conserved genome rearrangement in Delphacidae and discovery of new characteristics of atp8 and tRNA genes.

BACKGROUND: Nilaparvata lugens (the brown planthopper, BPH) and Laodelphax striatellus (the small brown planthopper, SBPH) are two of the most important pests of rice. Up to now, there was only one mitochondrial genome of rice planthopper has been sequenced and very few dependable information of mitochondria could be used for research on population genetics, phylogeographics and phylogenetic evolution of these pests. To get more valuable information from the mitochondria, we sequenced the complete mitochondrial genomes of BPH and SBPH. These two planthoppers were infected with two different functional Wolbachia (intracellular endosymbiont) strains (wLug and wStri). Since both mitochondria and Wolbachia are transmitted by cytoplasmic inheritance and it was difficult to separate them when purified the Wolbachia particles, concomitantly sequencing the genome of Wolbachia using next generation sequencing method, we also got nearly complete mitochondrial genome sequences of these two rice planthoppers. After gap closing, we present high quality and reliable complete mitochondrial genomes of these two planthoppers. RESULTS: The mitogenomes of N. lugens (BPH) and L. striatellus (SBPH) are 17, 619 bp and 16, 431 bp long with A + T contents of 76.95% and 77.17%, respectively. Both species have typical circular mitochondrial genomes that encode the complete set of 37 genes which are usually found in metazoans. However, the BPH mitogenome also possesses two additional copies of the trnC gene. In both mitochondrial genomes, the lengths of the atp8 gene were conspicuously shorter than that of all other known insect mitochondrial genomes (99 bp for BPH, 102 bp for SBPH). That two rearrangement regions (trnC-trnW and nad6-trnP-trnT) of mitochondrial genomes differing from other known insect were found in these two distantly related planthoppers revealed that the gene order of mitochondria might be conservative in Delphacidae. The large non-coding fragment (the A+T-rich region) putatively corresponding responsible for the control of replication and transcription of mitochondria contained a variable number of tandem repeats (VNTRs) block in different natural individuals of these two planthoppers. Comparison with a previously sequenced individual of SBPH revealed that the mitochondrial genetic variation within a species exists not only in the sequence and secondary structure of genes, but also in the gene order (the different location of trnH gene). CONCLUSION: The mitochondrial genome arrangement pattern found in planthoppers was involved in rearrangements of both tRNA genes and protein-coding genes (PCGs). Different species from different genera of Delphacidae possessing the same mitochondrial gene rearrangement suggests that gene rearrangements of mitochondrial genome probably occurred before the differentiation of this family. After comparatively analyzing the gene order of different species of Hemiptera, we propose that except for some specific taxonomical group (e.g. the whiteflies) the gene order might have diversified in family level of this order. The VNTRs detected in the control region might provide additional genetic markers for studying population genetics, individual difference and phylogeographics of planthoppers.

Multiple infections with Cardinium and two strains of Wolbachia in the spider mite Tetranychus phaselus Ehara: revealing new forces driving the spread of Wolbachia.

Cytoplasmic incompatibility (CI) has been proposed as a major mechanism by which certain strains of Wolbachia to invade and persist in host populations. However, mechanisms that underlie the invasion and persistence of non-CI strains are less well understood. Here, we established a spider mite Tetranychus phaselus population multiply infected by Cardinium as well as two distinct lineages of Wolbachia, designated wCon and wOri, to study the forces driving the spread of the non-CI strain of Wolbachia wOri. Interestingly, we found that wOri provided a longevity advantage to its female hosts under ideal conditions, making wOri stay longer in this population, and then being transmitted to more offspring. Furthermore, the lifespan of uninfected females was reduced when mated with multiple-infected males. As a result, the uninfected population is attenuated by the multiple-infected males. Thus, we infer that the host age effects of multiple infection may represent sufficient forces driving the spread of wOri through the host population.

The symbiosis phenotype and expression patterns of five nodule-specific genes of Astragalus sinicus under ammonium and salt stress conditions.

In previous works, we isolated 14 nodule-specific or nodule-enhanced genes from Astragalus sinicus by suppressive subtractive hybridization. In this study, we have further identified the expression patterns of five nodule-specific genes of A. sinicus under salt and ammonium stress. Transcription levels of genes tested were quantified by quantitative fluorescence real-time RT-PCR. Results showed that: (1) About 80 mM NaCl and all stress treatments containing (NH(4))(2)SO(4) significantly inhibited nitrogen-fixing capacity of inoculated plants. About 40 mM NaCl showed relative lighter inhibition. (2) Compare with positive control at normal conditions, the expressions of all genes were significantly reduced by all ammonium stress. (3) Under salt stress without exogenous nitrogen, transcription levels of AsIIA255 and AsE246 were significantly increased after treatment for 3 days. But expressions of AsG2411, AsIIC2512, and AsB2510 were suppressed by 80 mM NaCl and not significantly affected by 40 mM NaCl. (4) Under salt stress with exogenous nitrogen, expressions of AsG2411, AsIIC2512, AsB2510, and AsIIA255 were significantly suppressed. While, the transcription level of AsE246 under 80 mM NaCl containing 1 mM (NH(4))(2)SO(4) was still higher than that of positive control. The correlation of the expression profiles of three cysteine cluster protein (CCP) genes (AsG2411, AsIIC2512, AsIIA255) and one lipid transfer protein (LTP) gene (AsE246) with the nitrogen-fixing capacities of nodules in each treatments may explain the molecular mechanisms of their supposed functions in symbiosis and nitrogen-fixing process. Our results also implied that AsIIA255 and AsE246 might play a role in the response of A. sinicus to salt stress to facilitate the nitrogen-fixation process.

A novel nodule-enhanced gene encoding a putative universal stress protein from Astragalus sinicus.

A nodule-enhanced gene, AsD243, was identified from infected roots of Astragalus sinicus using suppressive subtractive hybridization (SSH). It encodes a 20-kD protein related to the bacterial universal stress protein family (Usp). Sequence analysis showed that AsD243 is highly similar to the bacterial MJ0577-type of ATP-binding Usp proteins, which have been proposed to function as a molecular switch. Expression analyses revealed that AsD243 was transcribed in all plant organs, and progressively during all stages of nodulation. Its transcripts increased significantly at 7 days after inoculation, which is 2 days later than the onset of leghemoglobin expression in A. sinicus nodules. AsD243 was expressed more strongly in mature roots than in young roots regardless of inoculation status. We suggest that the AsD243 may have other functions in plant processes besides nodulation.

Thirteen nodule-specific or nodule-enhanced genes encoding products homologous to cysteine cluster proteins or plant lipid transfer proteins are identified in Astragalus sinicus L. by suppressive subtractive hybridization.

Thirteen nodule-specific or nodule-enhanced genes have been revealed by suppressive subtractive hybridization (SSH) with two mRNA populations of infected and uninfected control roots of Astragalus sinicus. Eleven of them encode small polypeptides showing homology to cysteine cluster proteins (CCPs) that contain a putative signal peptide and conserved cysteine residues. Among these CCP-like genes, AsG257 codes for a homologue of the defensin 2 family and AsD255 contains a scorpion toxin-like domain at the C-terminus. Sequence analysis of a genomic AsD255 fragment which was isolated revealed that one intron separates the first exon encoding the signal peptide from the second exon encoding the cysteine cluster domain of this nodulin. Another two genes, AsE246 and AsIB259, encode two different products similar to lipid transfer proteins (LTPs). Virtual northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that the other genes except AsIB259 and AsC2411 were expressed exclusively in inoculated roots and that their expression was 2-4 d later than that of the leghaemoglobin (Lb) gene during nodule development. Transcription of AsIB259 was also detected in uninfected control roots but with a significant decline in expression and a temporal expression similar to Lb. AsC2411 had a basal expression in control roots identified by RT-PCR. Sequence alignment showed that the putative proteins AsE246 and AsIB259 show lower homology with LTPs from legumes than with those from other plants.

Symbiotic abilities of Sinorhizobium fredii with modified expression of purL.

Previous reports showed that a transposon-induced PurL- mutant of Sinorhizobium fredii induced pseudonodules on Glycine max and the addition of 5-aminoimidazole-4-carboxamide-riboside or adenine to the plant could not restore the mutant to establish effective symbiosis. To gain a better understanding of the impact of the purL gene on symbiosis formation, we measured the effect of modified expression of this gene on the symbiotic abilities of S. fredii on soybean (G. max). A 1.98-kb in-frame deletion mutant in the purL gene of S. fredii was constructed. Transcriptional modification of the purL gene was conducted using several promoters such as those of lac, nifH, nifQ, and fixN. It was found that reduced expression of purL gene or suitable symbiotic expression of purL (such as with the promoter nifH or nifQ) can efficiently establish symbiosis of S. fredii on G. max without the exogenous supplementation of any adenine or purine precursor; at least a minimal level of expression of purL is essential for effective symbiosis with soybean.