Protein interaction mapping reveals widespread targeting of development-related host transcription factors by phytoplasma effectors.

Phytoplasmas are pathogenic bacteria that reprogram plant host development for their own benefit. Previous studies have characterized a few different phytoplasma effector proteins that destabilize specific plant transcription factors. However, these are only a small fraction of the potential effectors used by phytoplasmas; therefore, the molecular mechanisms through which phytoplasmas modulate their hosts require further investigation. To obtain further insights into the phytoplasma infection mechanisms, we generated a protein-protein interaction network between a broad set of phytoplasma effectors and a large, unbiased collection of Arabidopsis thaliana transcription factors and transcriptional regulators. We found widespread, but specific, interactions between phytoplasma effectors and host transcription factors, especially those related to host developmental processes. In particular, many unrelated effectors target specific sets of TCP transcription factors, which regulate plant development and immunity. Comparison with other host-pathogen protein interaction networks shows that phytoplasma effectors have unusual targets, indicating that phytoplasmas have evolved a unique and unusual infection strategy. This study contributes a rich and solid data source that guides further investigations of the functions of individual effectors, as demonstrated for some herein. Moreover, the dataset provides insights into the underlying molecular mechanisms of phytoplasma infection.

Bimodular architecture of bacterial effector SAP05 that drives ubiquitin-independent targeted protein degradation.

In eukaryotes, targeted protein degradation (TPD) typically depends on a series of interactions among ubiquitin ligases that transfer ubiquitin molecules to substrates leading to degradation by the 26S proteasome. We previously identified that the bacterial effector protein SAP05 mediates ubiquitin-independent TPD. SAP05 forms a ternary complex via interactions with the von Willebrand Factor Type A (vWA) domain of the proteasomal ubiquitin receptor Rpn10 and the zinc-finger (ZnF) domains of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) and GATA BINDING FACTOR (GATA) transcription factors (TFs). This leads to direct TPD of the TFs by the 26S proteasome. Here, we report the crystal structures of the SAP05-Rpn10vWA complex at 2.17 Å resolution and of the SAP05-SPL5ZnF complex at 2.20 Å resolution. Structural analyses revealed that SAP05 displays a remarkable bimodular architecture with two distinct nonoverlapping surfaces, a "loop surface" with three protruding loops that form electrostatic interactions with ZnF, and a "sheet surface" featuring two ß-sheets, loops, and α-helices that establish polar interactions with vWA. SAP05 binding to ZnF TFs involves single amino acids responsible for multiple contacts, while SAP05 binding to vWA is more stable due to the necessity of multiple mutations to break the interaction. In addition, positioning of the SAP05 complex on the 26S proteasome points to a mechanism of protein degradation. Collectively, our findings demonstrate how a small bacterial bimodular protein can bypass the canonical ubiquitin-proteasome proteolysis pathway, enabling ubiquitin-independent TPD in eukaryotic cells. This knowledge holds significant potential for the creation of TPD technologies.

Hybridisation has shaped a recent radiation of grass-feeding aphids.

BACKGROUND: Aphids are common crop pests. These insects reproduce by facultative parthenogenesis involving several rounds of clonal reproduction interspersed with an occasional sexual cycle. Furthermore, clonal aphids give birth to live young that are already pregnant. These qualities enable rapid population growth and have facilitated the colonisation of crops globally. In several cases, so-called "super clones" have come to dominate agricultural systems. However, the extent to which the sexual stage of the aphid life cycle has shaped global pest populations has remained unclear, as have the origins of successful lineages. Here, we used chromosome-scale genome assemblies to disentangle the evolution of two global pests of cereals-the English (Sitobion avenae) and Indian (Sitobion miscanthi) grain aphids. RESULTS: Genome-wide divergence between S. avenae and S. miscanthi is low. Moreover, comparison of haplotype-resolved assemblies revealed that the S. miscanthi isolate used for genome sequencing is likely a hybrid, with one of its diploid genome copies closely related to S. avenae (~ 0.5% divergence) and the other substantially more divergent (> 1%). Population genomics analyses of UK and China grain aphids showed that S. avenae and S. miscanthi are part of a cryptic species complex with many highly differentiated lineages that predate the origins of agriculture. The complex consists of hybrid lineages that display a tangled history of hybridisation and genetic introgression. CONCLUSIONS: Our analyses reveal that hybridisation has substantially contributed to grain aphid diversity, and hence, to the evolutionary potential of this important pest species. Furthermore, we propose that aphids are particularly well placed to exploit hybridisation events via the rapid propagation of live-born "frozen hybrids" via asexual reproduction, increasing the likelihood of hybrid lineage formation.

Role of Acrostyle Cuticular Proteins in the Retention of an Aphid Salivary Effector.

To avoid the activation of plant defenses and ensure sustained feeding, aphids are assumed to use their mouthparts to deliver effectors into plant cells. A recent study has shown that effectors detected near feeding sites are differentially distributed in plant tissues. However, the precise process of effector delivery into specific plant compartments is unknown. The acrostyle, a cuticular organ located at the tip of maxillary stylets that transiently binds plant viruses via its stylin proteins, may participate in this specific delivery process. Here, we demonstrate that Mp10, a saliva effector released into the plant cytoplasm during aphid probing, binds to the acrostyles of Acyrthosiphon pisum and Myzus persicae. The effector probably interacts with Stylin-03 as a lowered Mp10-binding to the acrostyle was observed upon RNAi-mediated reduction in Stylin-03 production. In addition, Stylin-03 and Stylin-01 RNAi aphids exhibited changes in their feeding behavior as evidenced by electrical penetration graph experiments showing longer aphid probing behaviors associated with watery saliva release into the cytoplasm of plant cells. Taken together, these data demonstrate that the acrostyle also has effector binding capacity and supports its role in the delivery of aphid effectors into plant cells.

Diffusion and bulk flow of amino acids mediate calcium waves in plants.

In plants, a variety of stimuli trigger long-range calcium signals that travel rapidly along the vasculature to distal tissues via poorly understood mechanisms. Here, we use quantitative imaging and analysis to demonstrate that traveling calcium waves are mediated by diffusion and bulk flow of amino acid chemical messengers. We propose that wounding triggers release of amino acids that diffuse locally through the apoplast, activating the calcium-permeable channel GLUTAMATE RECEPTOR-LIKE 3.3 as they pass. Over long distances through the vasculature, the wound-triggered dynamics of a fluorescent tracer show that calcium waves are likely driven by bulk flow of a channel-activating chemical. We observed that multiple stimuli trigger calcium waves with similar dynamics, but calcium waves alone cannot initiate all systemic defense responses, suggesting that mobile chemical messengers are a core component of complex systemic signaling in plants.

Characterisation of the symbionts in the Mediterranean fruit fly gut.

Symbioses between bacteria and their insect hosts can range from loose associations through to obligate interdependence. While fundamental evolutionary insights have been gained from the in-depth study of obligate mutualisms, there is increasing interest in the evolutionary potential of flexible symbiotic associations between hosts and their gut microbiomes. Understanding relationships between microbes and hosts also offers the potential for exploitation for insect control. Here, we investigate the gut microbiome of a global agricultural pest, the Mediterranean fruit fly (Ceratitis capitata). We used 16S rRNA profiling to compare the gut microbiomes of laboratory and wild strains raised on different diets and from flies collected from various natural plant hosts. The results showed that medfly guts harbour a simple microbiome that is primarily determined by the larval diet. However, regardless of the laboratory diet or natural plant host on which flies were raised, Klebsiella spp. dominated medfly microbiomes and were resistant to removal by antibiotic treatment. We sequenced the genome of the dominant putative Klebsiella spp. ('Medkleb') isolated from the gut of the Toliman wild-type strain. Genome-wide ANI analysis placed Medkleb within the K. oxytoca / michiganensis group. Species level taxonomy for Medkleb was resolved using a mutli-locus phylogenetic approach - and molecular, sequence and phenotypic analyses all supported its identity as K. michiganensis. Medkleb has a genome size (5825435 bp) which is 1.6 standard deviations smaller than the mean genome size of free-living Klebsiella spp. Medkleb also lacks some genes involved in environmental sensing. Moreover, the Medkleb genome contains at least two recently acquired unique genomic islands as well as genes that encode pectinolytic enzymes capable of degrading plant cell walls. This may be advantageous given that the medfly diet includes unripe fruits containing high proportions of pectin. The results suggest that the medfly harbours a commensal gut bacterium that may have developed a mutualistic association with its host and provide nutritional benefits.

Parasitic modulation of host development by ubiquitin-independent protein degradation.

Certain obligate parasites induce complex and substantial phenotypic changes in their hosts in ways that favor their transmission to other trophic levels. However, the mechanisms underlying these changes remain largely unknown. Here we demonstrate how SAP05 protein effectors from insect-vectored plant pathogenic phytoplasmas take control of several plant developmental processes. These effectors simultaneously prolong the host lifespan and induce witches' broom-like proliferations of leaf and sterile shoots, organs colonized by phytoplasmas and vectors. SAP05 acts by mediating the concurrent degradation of SPL and GATA developmental regulators via a process that relies on hijacking the plant ubiquitin receptor RPN10 independent of substrate ubiquitination. RPN10 is highly conserved among eukaryotes, but SAP05 does not bind insect vector RPN10. A two-amino-acid substitution within plant RPN10 generates a functional variant that is resistant to SAP05 activities. Therefore, one effector protein enables obligate parasitic phytoplasmas to induce a plethora of developmental phenotypes in their hosts.

Plant pathogens convergently evolved to counteract redundant nodes of an NLR immune receptor network.

In plants, nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins can form receptor networks to confer hypersensitive cell death and innate immunity. One class of NLRs, known as NLR required for cell death (NRCs), are central nodes in a complex network that protects against multiple pathogens and comprises up to half of the NLRome of solanaceous plants. Given the prevalence of this NLR network, we hypothesised that pathogens convergently evolved to secrete effectors that target NRC activities. To test this, we screened a library of 165 bacterial, oomycete, nematode, and aphid effectors for their capacity to suppress the cell death response triggered by the NRC-dependent disease resistance proteins Prf and Rpi-blb2. Among 5 of the identified suppressors, 1 cyst nematode protein and 1 oomycete protein suppress the activity of autoimmune mutants of NRC2 and NRC3, but not NRC4, indicating that they specifically counteract a subset of NRC proteins independently of their sensor NLR partners. Whereas the cyst nematode effector SPRYSEC15 binds the nucleotide-binding domain of NRC2 and NRC3, the oomycete effector AVRcap1b suppresses the response of these NRCs via the membrane trafficking-associated protein NbTOL9a (Target of Myb 1-like protein 9a). We conclude that plant pathogens have evolved to counteract central nodes of the NRC immune receptor network through different mechanisms. Coevolution with pathogen effectors may have driven NRC diversification into functionally redundant nodes in a massively expanded NLR network.

Spotlight on the Roles of Whitefly Effectors in Insect-Plant Interactions.

The Bemisia tabaci species complex (whitefly) causes enormous agricultural losses. These phloem-feeding insects induce feeding damage and transmit a wide range of dangerous plant viruses. Whiteflies colonize a broad range of plant species that appear to be poorly defended against these insects. Substantial research has begun to unravel how phloem feeders modulate plant processes, such as defense pathways, and the central roles of effector proteins, which are deposited into the plant along with the saliva during feeding. Here, we review the current literature on whitefly effectors in light of what is known about the effectors of phloem-feeding insects in general. Further analysis of these effectors may improve our understanding of how these insects establish compatible interactions with plants, whereas the subsequent identification of plant defense processes could lead to improved crop resistance to insects. We focus on the core concepts that define the effectors of phloem-feeding insects, such as the criteria used to identify candidate effectors in sequence-mining pipelines and screens used to analyze the potential roles of these effectors and their targets in planta. We discuss aspects of whitefly effector research that require further exploration, including where effectors localize when injected into plant tissues, whether the effectors target plant processes beyond defense pathways, and the properties of effectors in other insect excretions such as honeydew. Finally, we provide an overview of open issues and how they might be addressed.

Chromosome-Scale Genome Assemblies of Aphids Reveal Extensively Rearranged Autosomes and Long-Term Conservation of the X Chromosome.

Chromosome rearrangements are arguably the most dramatic type of mutations, often leading to rapid evolution and speciation. However, chromosome dynamics have only been studied at the sequence level in a small number of model systems. In insects, Diptera and Lepidoptera have conserved genome structure at the scale of whole chromosomes or chromosome arms. Whether this reflects the diversity of insect genome evolution is questionable given that many species exhibit rapid karyotype evolution. Here, we investigate chromosome evolution in aphids-an important group of hemipteran plant pests-using newly generated chromosome-scale genome assemblies of the green peach aphid (Myzus persicae) and the pea aphid (Acyrthosiphon pisum), and a previously published assembly of the corn-leaf aphid (Rhopalosiphum maidis). We find that aphid autosomes have undergone dramatic reorganization over the last 30 My, to the extent that chromosome homology cannot be determined between aphids from the tribes Macrosiphini (Myzus persicae and Acyrthosiphon pisum) and Aphidini (Rhopalosiphum maidis). In contrast, gene content of the aphid sex (X) chromosome remained unchanged despite rapid sequence evolution, low gene expression, and high transposable element load. To test whether rapid evolution of genome structure is a hallmark of Hemiptera, we compared our aphid assemblies with chromosome-scale assemblies of two blood-feeding Hemiptera (Rhodnius prolixus and Triatoma rubrofasciata). Despite being more diverged, the blood-feeding hemipterans have conserved synteny. The exceptional rate of structural evolution of aphid autosomes renders them an important emerging model system for studying the role of large-scale genome rearrangements in evolution.

A chromosome-level genome assembly of the woolly apple aphid, Eriosoma lanigerum Hausmann (Hemiptera: Aphididae).

Woolly apple aphid (WAA, Eriosoma lanigerum Hausmann) (Hemiptera: Aphididae) is a major pest of apple trees (Malus domestica, order Rosales) and is critical to the economics of the apple industry in most parts of the world. Here, we generated a chromosome-level genome assembly of WAA-representing the first genome sequence from the aphid subfamily Eriosomatinae-using a combination of 10X Genomics linked-reads and in vivo Hi-C data. The final genome assembly is 327 Mb, with 91% of the assembled sequences anchored into six chromosomes. The contig and scaffold N50 values are 158 kb and 71 Mb, respectively, and we predicted a total of 28,186 protein-coding genes. The assembly is highly complete, including 97% of conserved arthropod single-copy orthologues based on Benchmarking Universal Single-Copy Orthologs (busco) analysis. Phylogenomic analysis of WAA and nine previously published aphid genomes, spanning four aphid tribes and three subfamilies, reveals that the tribe Eriosomatini (represented by WAA) is recovered as a sister group to Aphidini + Macrosiphini (subfamily Aphidinae). We identified syntenic blocks of genes between our WAA assembly and the genomes of other aphid species and find that two WAA chromosomes (El5 and El6) map to the conserved Macrosiphini and Aphidini X chromosome. Our high-quality WAA genome assembly and annotation provides a valuable resource for research in a broad range of areas such as comparative and population genomics, insect-plant interactions and pest resistance management.

Genome Sequence of the Banana Aphid, Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae) and Its Symbionts.

The banana aphid, Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae), is a major pest of cultivated bananas (Musa spp., order Zingiberales), primarily due to its role as a vector of Banana bunchy top virus (BBTV), the most severe viral disease of banana worldwide. Here, we generated a highly complete genome assembly of P. nigronervosa using a single PCR-free Illumina sequencing library. Using the same sequence data, we also generated complete genome assemblies of the P. nigronervosa symbiotic bacteria Buchnera aphidicola and Wolbachia To improve our initial assembly of P. nigronervosa we developed a k-mer based deduplication pipeline to remove genomic scaffolds derived from the assembly of haplotigs (allelic variants assembled as separate scaffolds). To demonstrate the usefulness of this pipeline, we applied it to the recently generated assembly of the aphid Myzus cerasi, reducing the duplication of conserved BUSCO genes by 25%. Phylogenomic analysis of P. nigronervosa, our improved M. cerasi assembly, and seven previously published aphid genomes, spanning three aphid tribes and two subfamilies, reveals that P. nigronervosa falls within the tribe Macrosiphini, but is an outgroup to other Macrosiphini sequenced so far. As such, the genomic resources reported here will be useful for understanding both the evolution of Macrosphini and for the study of P. nigronervosa. Furthermore, our approach using low cost, high-quality, Illumina short-reads to generate complete genome assemblies of understudied aphid species will help to fill in genomic black spots in the diverse aphid tree of life.

An Aphid-Secreted Salivary Protease Activates Plant Defense in Phloem.

Recent studies have reported that aphids facilitate their colonization of host plants by secreting salivary proteins into host tissues during their initial probing and feeding. Some of these salivary proteins elicit plant defenses, but the molecular and biochemical mechanisms that underlie the activation of phloem-localized resistance remain poorly understood. The aphid Myzus persicae, which is a generalized phloem-sucking pest, encompasses a number of lineages that are associated with and adapted to specific host plant species. The current study found that a cysteine protease Cathepsin B3 (CathB3), and the associated gene CathB3, was upregulated in the salivary glands and saliva of aphids from a non-tobacco-adapted (NTA) aphid lineage, when compared to those of a tobacco-adapted lineage. Furthermore, the knockdown of CathB3 improved the performance of NTA lineages on tobacco, and the propeptide domain of CathB3 was found to bind to tobacco cytoplasmic kinase ENHANCED DISEASE RESISTANCE 1-like (EDR1-like), which triggers the accumulation of reactive oxygen species in tobacco phloem, thereby suppressing both phloem feeding and colonization by NTA lineages. These findings reveal a novel function for a cathepsin-type protease in aphid saliva that elicits effective host plant defenses and warranted the theory of host specialization for generalist aphids.

Complete Genome Sequence of "Candidatus Phytoplasma asteris" RP166, a Plant Pathogen Associated with Rapeseed Phyllody Disease in Poland.

The complete genome sequence of "Candidatus Phytoplasma asteris" RP166, which consists of one 829,546-bp circular chromosome, is presented in this work. This bacterium is associated with rapeseed phyllody disease in Poland and belongs to the 16SrI-B (i.e., aster yellows) group.

Species Boundaries and Molecular Markers for the Classification of 16SrI Phytoplasmas Inferred by Genome Analysis.

Phytoplasmas are plant-pathogenic bacteria that impact agriculture worldwide. The commonly adopted classification system for phytoplasmas is based on the restriction fragment length polymorphism (RFLP) analysis of their 16S rRNA genes. With the increased availability of phytoplasma genome sequences, the classification system can now be refined. This work examined 11 strains in the 16SrI group within the genus 'Candidatus Phytoplasma' and investigated the possible species boundaries. We confirmed that the RFLP classification method is problematic due to intragenomic variation of the 16S rRNA genes and uneven weighing of different nucleotide positions. Importantly, our results based on the molecular phylogeny, differentiations in chromosomal segments and gene content, and divergence in homologous sequences, all supported that these strains may be classified into multiple operational taxonomic units (OTUs) equivalent to species. Strains assigned to the same OTU share >97% genome-wide average nucleotide identity (ANI) and >78% of their protein-coding genes. In comparison, strains assigned to different OTUs share < 94% ANI and < 75% of their genes. Reduction in homologous recombination between OTUs is one possible explanation for the discontinuity in genome similarities, and these findings supported the proposal that 95% ANI could serve as a cutoff for distinguishing species in bacteria. Additionally, critical examination of these results and the raw sequencing reads led to the identification of one genome that was presumably mis-assembled by combining two sequencing libraries built from phytoplasmas belonging to different OTUs. This finding provided a cautionary tale for working on uncultivated bacteria. Based on the new understanding of phytoplasma divergence and the current genome availability, we developed five molecular markers that could be used for multilocus sequence analysis (MLSA). By selecting markers that are short yet highly informative, and are distributed evenly across the chromosome, these markers provided a cost-effective system that is robust against recombination. Finally, examination of the effector gene distribution further confirmed the rapid gains and losses of these genes, as well as the involvement of potential mobile units (PMUs) in their molecular evolution. Future improvements on the taxon sampling of phytoplasma genomes will allow further expansions of similar analysis, and thus contribute to phytoplasma taxonomy and diagnostics.

Bacterial Vector-Borne Plant Diseases: Unanswered Questions and Future Directions.

Vector-borne plant diseases have significant ecological and economic impacts, affecting farm profitability and forest composition throughout the world. Bacterial vector-borne pathogens have evolved sophisticated strategies to interact with their hemipteran insect vectors and plant hosts. These pathogens reside in plant vascular tissue, and their study represents an excellent opportunity to uncover novel biological mechanisms regulating intracellular pathogenesis and to contribute to the control of some of the world's most invasive emerging diseases. In this perspective, we highlight recent advances and major unanswered questions in the realm of bacterial vector-borne disease, focusing on liberibacters, phytoplasmas, spiroplasmas, and Xylella fastidiosa.

An aphid RNA transcript migrates systemically within plants and is a virulence factor.

Aphids are sap-feeding insects that colonize a broad range of plant species and often cause feeding damage and transmit plant pathogens, including bacteria, viruses, and viroids. These insects feed from the plant vascular tissue, predominantly the phloem. However, it remains largely unknown how aphids, and other sap-feeding insects, establish intimate long-term interactions with plants. To identify aphid virulence factors, we took advantage of the ability of the green peach aphid Myzus persicae to colonize divergent plant species. We found that a M. persicae clone of near-identical females established stable colonies on nine plant species of five representative plant eudicot and monocot families that span the angiosperm phylogeny. Members of the novel aphid gene family Ya are differentially expressed in aphids on the nine plant species and are coregulated and organized as tandem repeats in aphid genomes. Aphids translocate Ya transcripts into plants, and some transcripts migrate to distal leaves within several plant species. RNAi-mediated knockdown of Ya genes reduces M. persicae fecundity, and M. persicae produces more progeny on transgenic plants that heterologously produce one of the systemically migrating Ya transcripts as a long noncoding (lnc) RNA. Taken together, our findings show that beyond a range of pathogens, M. persicae aphids translocate their own transcripts into plants, including a Ya lncRNA that migrates to distal locations within plants, promotes aphid fecundity, and is a member of a previously undescribed host-responsive aphid gene family that operate as virulence factors.

Phytoplasma SAP11 effector destabilization of TCP transcription factors differentially impact development and defence of Arabidopsis versus maize.

Phytoplasmas are insect-transmitted bacterial pathogens that colonize a wide range of plant species, including vegetable and cereal crops, and herbaceous and woody ornamentals. Phytoplasma-infected plants often show dramatic symptoms, including proliferation of shoots (witch's brooms), changes in leaf shapes and production of green sterile flowers (phyllody). Aster Yellows phytoplasma Witches' Broom (AY-WB) infects dicots and its effector, secreted AYWB protein 11 (SAP11), was shown to be responsible for the induction of shoot proliferation and leaf shape changes of plants. SAP11 acts by destabilizing TEOSINTE BRANCHED 1-CYCLOIDEA-PROLIFERATING CELL FACTOR (TCP) transcription factors, particularly the class II TCPs of the CYCLOIDEA/TEOSINTE BRANCHED 1 (CYC/TB1) and CINCINNATA (CIN)-TCP clades. SAP11 homologs are also present in phytoplasmas that cause economic yield losses in monocot crops, such as maize, wheat and coconut. Here we show that a SAP11 homolog of Maize Bushy Stunt Phytoplasma (MBSP), which has a range primarily restricted to maize, destabilizes specifically TB1/CYC TCPs. SAP11MBSP and SAP11AYWB both induce axillary branching and SAP11AYWB also alters leaf development of Arabidopsis thaliana and maize. However, only in maize, SAP11MBSP prevents female inflorescence development, phenocopying maize tb1 lines, whereas SAP11AYWB prevents male inflorescence development and induces feminization of tassels. SAP11AYWB promotes fecundity of the AY-WB leafhopper vector on A. thaliana and modulates the expression of A. thaliana leaf defence response genes that are induced by this leafhopper, in contrast to SAP11MBSP. Neither of the SAP11 effectors promote fecundity of AY-WB and MBSP leafhopper vectors on maize. These data provide evidence that class II TCPs have overlapping but also distinct roles in regulating development and defence in a dicot and a monocot plant species that is likely to shape SAP11 effector evolution depending on the phytoplasma host range.

Sex-specific changes in the aphid DNA methylation landscape.

Aphids present an ideal system to study epigenetics as they can produce diverse, but genetically identical, morphs in response to environmental stimuli. Here, using whole genome bisulphite sequencing and transcriptome sequencing of the green peach aphid (Myzus persicae), we present the first detailed analysis of cytosine methylation in an aphid and investigate differences in the methylation and transcriptional landscapes of male and asexual female morphs. We found that methylation primarily occurs in a CG dinucleotide (CpG) context and that exons are highly enriched for methylated CpGs, particularly at the 3' end of genes. Methylation is positively associated with gene expression, and methylated genes are more stably expressed than unmethylated genes. Male and asexual female morphs have distinct methylation profiles. Strikingly, these profiles are divergent between the sex chromosome and the autosomes; autosomal genes are hypomethylated in males compared to asexual females, whereas genes belonging to the sex chromosome, which is haploid in males, are hypermethylated. Overall, we found correlated changes in methylation and gene expression between males and asexual females, and this correlation was particularly strong for genes located on the sex chromosome. Our results suggest that differential methylation of sex-biased genes plays a role in aphid sexual differentiation.

The genome of 'Candidatus Phytoplasma solani' strain SA-1 is highly dynamic and prone to adopting foreign sequences.

Bacteria of the genus 'Candidatus Phytoplasma' are uncultivated intracellular plant pathogens transmitted by phloem-feeding insects. They have small genomes lacking genes for essential metabolites, which they acquire from either plant or insect hosts. Nonetheless, some phytoplasmas, such as 'Ca. P. solani', have broad plant host range and are transmitted by several polyphagous insect species. To understand better how these obligate symbionts can colonize such a wide range of hosts, the genome of 'Ca. P. solani' strain SA-1 was sequenced from infected periwinkle via a metagenomics approach. The de novo assembly generated a draft genome with 19 contigs totalling 821,322bp, which corresponded to more than 80% of the estimated genome size. Further completion of the genome was challenging due to the high occurrence of repetitive sequences. The majority of repeats consisted of gene arrangements characteristic of phytoplasma potential mobile units (PMUs). These regions showed variation in gene orders intermixed with genes of unknown functions and lack of similarity to other phytoplasma genes, suggesting that they were prone to rearrangements and acquisition of new sequences via recombination. The availability of this high-quality draft genome also provided a foundation for genome-scale genotypic analysis (e.g., average nucleotide identity and average amino acid identity) and molecular phylogenetic analysis. Phylogenetic analyses provided evidence of horizontal transfer for PMU-like elements from various phytoplasmas, including distantly related ones. The 'Ca. P. solani' SA-1 genome also contained putative secreted protein/effector genes, including a homologue of SAP11, found in many other phytoplasma species.