The transmission process of vector-borne viruses is categorized by two features: (1) the time period required by the vector for acquisition of the virus and inoculation of the virus, and (2) the retention time of viral particles in the vector (Ng and Falk, 2006). Based on these features, virus-vector relationships can be categorized as non-persistent, semi-persistent, or persistent. For non-persistent viruses, transmission can occur within minutes of acquiring the viral particles (virions) and particles are retained in the stylet or in the alimentary canal of the insect (Ng and Falk, 2006; Uzest et al., 2007; Whitfield et al., 2015). Viral particles can be lost quickly in this transmission mode and multiple encounters with infected plants are required for vectors to remain viruliferous (Ng and Falk, 2006). Semi-persistent retention of virions can last for days and retention sites are found in the alimentary canal or gut lumen of the insect for the majority of these viruses (Chen et al., 2011; Ng and Zhou, 2015). For semi-persistent relationships, feeding for hours to days is required to acquire the virus and if acquisition occurs during vector immature stages, infectivity is lost after each molt. Finally for persistent associations, vectors remain infective until death after a single encounter with an infected plant. Long feeding periods (hours to days) are required for acquisition of persistent viruses by vectors.

Persistence of vector-borne bacteria varies according to plant-tissue specialization. Xylella fastidiosa, the only known vector-borne xylem specialist, has a semi-persistent association with its vectors (Table 1). Dozens of crops and native plants are hosts for X. fastidiosa and a diverse array of vectors transmits the pathogen compared to other species of vector-borne bacteria (Redak et al., 2004). The ability to utilize diverse plant and vector species may be due to X. fastidiosa’s semi-persistent relationship with insects. For example, semi-persistent bacteria may be more easily acquired and transmitted by vectors to diverse host species during pre-feeding and host finding behavior. In contrast, all known phloem-limited bacteria appear to establish persistent associations with their respective vectors (Table 1). Persistence of phloem specialists may be due to the intracellular relationship they share with plant and insect hosts. However, conclusion about tissue trophisms may be premature, as only one vector-borne xylem specialists is known so far.

The interactions plant viruses share with their insect vectors can either be “non-circulative” or “circulative.” In non-circulative interactions, the virus does not enter the insect body as part of the transmission process and the virus particles are retained in the stylet or the foregut region (Ng and Zhou, 2015). Viruses that are transmitted in a circulative manner in contrast, pass beyond the foregut into the insect intestine and enter the body as part of the transmission process. Circulative viruses can be retained for the life of the insect vector (Gray et al., 2014). For vector-borne bacteria both non-circulative and circulative relationships exist among pathogen–vector interactions (Table 1); and like persistence, relationships correlate with plant-tissue specialization of the pathogen. For example, the xylem colonizer, X. fastidiosa, is non-circulative, while all known phloem colonizers interact in a circulative manner with vectors (Table 1).

Differences in pathogen location within vectors may be explained by ancestral origins (Nadarasah and Stavrinides, 2011). In one scenario, bacteria pre-adapted to plant environments may have evolved to use insects as alternative hosts. Alternatively, insect pathogens or symbionts, pre-adapted to thrive in hemipterans, may have found an additional niche in plants (Nadarasah and Stavrinides, 2011). X. fastidiosa is most closely related to the genus Xanthomonas (Table 1). Members of Xanthomonas are exclusively plant-associated and commonly plant pathogens. Inability to cross insect membranes may be due to the fact that X. fastidiosa has evolved to be restricted to dead cells of the plant (xylem). The ability to cross plant cellular membranes may have been lost from its genetic arsenal over time. Liberibacters also are related to plant pathogens as a member of the family Rhizobiaceae, yet liberibacters have circulative relationships with insect vectors. A more striking phylogenetic observation for liberibacters is that many members of the Rhizobiaceae, have intracellular associations with hosts as pathogens and symbionts (insect and plant hosts; genera Bradyrhizobium, Bartonella, Brucella, and Afipia; Jagoueix et al., 1994). This trend may explain the origin of the circulative associations of liberibacters with their vectors. As microbiome projects for hemipterans expand, the relationship among these bacteria and the traits responsible for interactions inside the insect will likely be revealed.

Circulative viral pathogens can either circulate through the insect vector’s body without reproducing, in which case they are described as “non-propagative,” or they can circulate and multiply within the insect vector, in which case they are described as “propagative.” In the latter case, the vector serves as an alternative host for the plant pathogen (Nadarasah and Stavrinides, 2011). Typically, the vector acquires the plant pathogen by feeding on infected plants. Once inside the insect body, the virus crosses intestinal barriers, internal organs, and visceral muscles, and can be found throughout the hemolymph (Hogenhout et al., 2008a; Orlovskis et al., 2015). From the hemolymph the virus must spread to the salivary glands before the vector can subsequently transmit the pathogen to a new plant host. Only a few families of vector-borne plant viruses have propagative relationships with vectors. These families include Rhabdoviridae, Reoviridae, and Bunyaviridae (Hogenhout et al., 2008a; Ammar et al., 2009; Whitfield et al., 2015).

All described vector-borne bacteria utilize their insect vectors as alternative hosts, and are thus considered propagative (Bove and Garnier, 2003; Orlovskis et al., 2015). Vector-borne bacteria can propagate extracellularly (between host cells) or intracellularly (within host cells; Table 1). For example, the xylem colonizer, X. fastidiosa, propagates extracellularly within the vector and is non-circulative. This is in contrast to all vector-borne viruses, which can only be propagative and circulative, as they are all parasites of the cellular replication machinery. It is assumed all phloem colonizers propagate intracellularly within their vectors as they are found in diverse tissues and hemolymph (Table 1). However, detailed intracellular propagation of bacteria is not easily studied and the current knowledge may reflect methodological limitations for evaluating bacterial replication in different insect organs and cavities. Specific mechanisms mediating insect recognition, attachment, and multiplication in organs are also not yet clear, and seem to be unique for each bacteria–vector interaction.

The only known xylem-limited bacterial pathogen that is also transmitted by hemipteran vectors is X. fastidiosa. X. fastidiosa (class Gammaproteobacteria) has a very wide host range, colonizing and causing disease in grapes (Pierce’s disease), citrus (citrus variegated chlorosis), olives (leaf scorch), almonds (leaf scorch), and several other plant species (Chatterjee et al., 2008a; Saponari et al., 2014; Almeida and Nuney, 2015). Because of its economic importance, X. fastidiosa was the first bacterial plant pathogen genome to be completely sequenced (Simpson et al., 2000). The genome of X. fastidiosa is ~2.7 Mb, half the size of its closest relatives (Xanthomonas group; Table 1). X. fastidiosa is transmitted in a non-circulative manner by a diverse set of xylem-feeding hemipterans including members from the superfamilies Membracoidea, Cercopoidea, and Cicadoidea (PaiãO et al., 1996; Purcell and Hopkins, 1996; Saponari et al., 2014). Experimental evidence suggests that a wide range of additional xylem feeding hemipterans are potential vectors, however, efficiency of transmission may vary depending on the vector species (Redak et al., 2004). The planthoppers Homalodisca vitripennis and Graphocephala atropunctata are the two most well studied vectors of X. fastidiosa. Knowledge of these two vectors has been used to model the relationship X. fastidiosa share with vectors in general (Chatterjee et al., 2008b; Backus and Morgan, 2011; Rapicavoli et al., 2015).

Numerous research tools have been developed to study X. fastidiosa, including in vitro culture techniques, transformation, and bacterial strain mutants (Killiny et al., 2012; Purcell, 2013; Webster et al., 2014; Rapicavoli et al., 2015). These tools have facilitated the dissection of many genetic components involved in pathogenicity. X. fastidiosa colonize and propagate extracellularly in the plant and insect (Chatterjee et al., 2008a), with some overlapping mechanisms. X. fastidiosa uses a cell-to-cell signaling sensor (RpfC), which acts as a negative regulator. This signaling system is mediated by diffusible signaling factors (DSF) in order to modulate different aspects of behavior in a population dependent manner (i.e., quorum sensing). DSF is secreted into the extracellular environment, activating motility, biofilm formation, and virulence mechanisms when a threshold concentration is reached outside the cell (Chatterjee et al., 2008a). Aspects of X. fastidiosa colonization that are dependent on quorum sensing include the production of toxins, extracellular polysaccharides (EPS), adhesins, and hemaglutinins (Table 2) (Chatterjee et al., 2008b; Nascimento et al., 2016). Inside the insect, the bacteria do not invade the epithelial gut, hemolymph, or salivary glands, and are retained in the alimentary canal. Transmission can occur within minutes of acquisition (Killiny et al., 2012). Recently it was determined that lipopolysaccharide (LPS), the outermost layer of sugar polymers surrounding Gram-negative bacteria, is critical for attachment to the vector and subsequent transmission (Table 2) (Rapicavoli et al., 2015).

In plants, X. fastidiosa relies on bacterial multiplication, attachment, and dispersion into neighboring vessels to colonize the xylem (Chatterjee et al., 2008b; Nascimento et al., 2016). Phytotoxicity during early stages of infection is associated with the lipase/esterase effector LesA, a type II secreted enzyme produced abundantly in culture (Nascimento et al., 2016). By degrading plant cell walls, nutrients are acquired and the bacteria are able to disperse throughout the plant (Purcell, 2013; Fatima and Senthil-Kumar, 2015). Degradation of vascular plant tissue requires the combined action of multiple enzymes, such as β-1,4 endoglucanases, xylanases, xylosidases, and polygalacturonases (Purcell, 2013; Fatima and Senthil-Kumar, 2015). X. fastidiosa mutants impaired in polygalacturonases enzyme (PglA) production, lack pathogenicity, and systemic movement in the plant (Table 2) (Roper et al., 2007). Given the reduced nutritional content of the xylem and the extracellular location of X. fastidiosa, many differences may exist for pathogenicity strategies among xylem and phloem colonizers.

Diverse phylogenetic groups converge in phloem specialization and hemipteran transmission and it is hypothesized that those traits have been acquired independently multiple times over the course of bacteria evolution (Orlovskis et al., 2015). The majority of phytoplasmas (class Mollicutes) and liberibacters (class Alphaproteobacteria) are vector-borne phytopathogens (Bressan, 2014; Fagen et al., 2014a). For other groups, such as spiroplasmas (class Mollicutes), only some species are phytopathogens. Despite this diversity, all known phloem-limited vector-borne bacteria appear to colonize both the insect vector and the plant host intracellularly (Orlovskis et al., 2015). The bacteria cross the gut barrier and circulate in the vector body, eventually reaching the hemolymph, and salivary glands (Table 1) (Gasparich, 2010). The journey to the salivary glands requires a latent period, ranging from days to months, before transmission can occur (Thebaud et al., 2009). Here, we will discuss three examples of phylogenetic groups containing vector-borne bacteria: spiroplasmas, phytoplasmas, and liberibacters.

Spiroplasma spp. have a distinctive helical morphology and use pili–like structures to move in a corkscrew-like motion (Ammar et al., 2004). They are classified as Mollicutes as they lack a cell wall. Spiroplasmas share diverse relationships with plant and insect hosts spanning pathogenic, commensal, and mutualistic interactions (Ammar et al., 2004). Most are associated with diverse insect orders, such as Hymenoptera, Coleoptera, Diptera, Lepidoptera, and Hemiptera (Gasparich, 2010). However, there are three phytopathogenic spiroplasmas that are also transmitted by leafhoppers (Cicadellidae): Spiroplasma citri, S. kunkelii, and S. phoeniceum (Table 1) (Gasparich, 2010). S. citri, the causal agent of citrus stubborn, was the first vector-borne bacteria to be cultured. It was first discovered in 1970 and culture methods were developed shortly after this (Bove and Garnier, 2003). Cultivation of spiroplasmas is not trivial, as it requires complex media enriched with cholesterol and fatty acids. Tools and information derived for spiroplasmas culture methods have served as references for attempts to culture phytoplasmas and liberibacter.

After acquisition, spiroplasmas adhere to receptors in the lumen of the insect midgut, where endocytosis occurs (Fletcher et al., 1998; Gasparich, 2010). Intracellular vesicular transport mediates migration to the hemolymph and exocytosis (Fletcher et al., 1998). Once inside the hemolymph, the bacteria are transported throughout the insect body, eventually reaching the salivary glands after additional intracellular crossings (Fletcher et al., 1998). Currently, the specific insect receptors/factors mediating the journey inside the vector remain unknown for spiroplasmas. However, several potential proteins required for insect attachment have been identified using S. citri mutants impaired in insect transmission and with S. citri strains that have lost insect attachment properties after multiple in vitro cultivations (Ye et al., 1997; Fletcher et al., 1998; Berho et al., 2006; Mutaqin et al., 2011) (Table 2).

One of the first approaches developed to study bacterial protein–insect interactions was the use of leafhopper (C. tenellus) monolayer cell culture assays with spiroplasmas. In this technique, researchers exposed insect cells (CT1) in vitro to S. citri. After exposure, electron microscopy (Wayadande and Fletcher, 1998) or immunofluorescence assays (Labroussaa et al., 2010) were used to evaluate bacterial phenotypes. Numerous candidate attachment proteins have been identified in this way, including P58, SARP, and the plasmid-borne protein P32 (Table 2) (Wayadande and Fletcher, 1998; Berg et al., 2001). Another very abundant membrane protein of S. citri that has been implicated in transmission is spiralin. S. citri mutants compromised in spiralin production exhibit reduced transmission by the vector, Circulifer haematoceps (Table 2) (Gasparich, 2010). This suggests that spiralin may mediate pathogen interactions within the insect vector, though specific mechanisms remain unknown.

Phytoplasmas are another category of the Mollicutes that depend on insect vectors for transmission (Table 1), but unlike Spiroplasma, they have pleomorphic shapes and are very difficult to culture (Contaldo et al., 2012). Phytoplasmas are a diverse monophyletic group, with more than 30 “Candidatus Phytoplasma” species described and 100s of subgroups (Hogenhout et al., 2008b). As a taxon they have a wide host range, infecting more than 800 different plant species (Hogenhout et al., 2008b), but individual strains have highly restricted insect and plant hosts. Collectively, more than 1000 plant diseases are caused by phytoplasmas that are transmitted by leafhoppers and, to a lesser extent, a few other hemipterans (Mitchel, 2004; Weintraub and Beanland, 2006).

Phytoplasmas have the smallest genomes of all described phytopathogenic bacteria, averaging ~0.7 Mb with a low G+C content, high number of repetitive regions, and interesting variability in genome features across the taxon (Table 2) (Kube et al., 2012, 2014). At least six types of ATP-binding cassette (ABC) transporters are conserved in the evaluated genomes. ABC transporters shuttle metabolites across bacterial membranes, and are predicted to allow nutrient and metabolite uptake from the host. Other common features include a superoxide dismutase enzyme (SOD), possibly used to counteract reactive oxygen species produced by hosts, and a protease (HflB), which is a virulence factor for ‘Candidatus Phytoplasma mali (Wang et al., 2014). Recently a conserved Mollicutes adhesion motif (MAM) was identified in the Onion Yellow Phytoplasma genome. This candidate protein (P38) interacts with crude insect extracts and weakly with plants extracts (Table 2) (Neriya et al., 2014), however, specific host targets are unknown.

Phytoplasmas also encode translocase SecA, part of the Type II secretion system for bacteria. This secretion system allows the delivery of functionally distinct proteins with a characteristic signal peptide at the n-terminal to the bacterial membrane. Because phytoplasmas have a single membrane, after the signal peptide is cleaved the proteins are released into the host environment (secreted). Secreted phytoplasma proteins can alter host functions and act as effectors (Bai et al., 2009). A single phytoplasma genome can encodes over 50 secreted proteins (SAP’s), however, the function of each one during host colonization and propagation is only known for a few (Bai et al., 2009). SAP effectors often alter host function by manipulating plant hormone homeostasis. For example, the effector TENGU inhibits auxin-related pathways leading to a dwarf plant phenotype and floral sterility (Table 2) (Minato et al., 2014). Further, Arabidopsis transgenic lines expressing SAP11, produce less jasmonic acid (JA) compared to controls (Table 2) (Sugio et al., 2014). This leads to abnormal vegetative growth and increased fecundity for leafhopper vectors on infected plants (Lu et al., 2014b). SAP effectors can also modulate pathogenicity through changes in development. SAP54/PHYL interacts with floral transcription factors and promotes degradation of the MADS-box proteins. MADS-box proteins are critical for floral meristem development and plants expressing SAP54/PHYL flower abnormally (Table 1) (MacLean et al., 2014; Maejima et al., 2014).

A second group of proteins delivered by the Sec-secretion system are the immunodominant membrane proteins (IMPs), which remain anchored and decorate the external membrane of phytoplasmas. IMPs are unique for phytoplasmas and are categorized into three subgroups depending on whether the n- or c- terminal side of the protein is exposed extracellularly (Amp, IdpA, or Imp; Kakizawa et al., 2006). When a monoclonal anti-AMP from “Candidatus Phytoplasma asteris” Chrysanthemums Yellows strain (CPY) was fed to the leafhopper vector, internalization of the phytoplasma and transmission efficiency was reduced. These results imply that anti-Amp impedes attachment of the bacteria in the vector gut (Table 2) (Rashidi et al., 2015).

The genus Liberibacter spp. contains six species of phloem-limited bacteria (Haapalainen, 2014): “Ca. Liberibacter africanus,” “Ca. Liberibacter americanus,” “Ca. Liberibacter asiaticus,” “Ca. Liberibacter solanacearum,” “Ca. Liberibacter europaeus,” and Liberibacter crescens. “Ca. Liberibacter africanus,” “Ca. Liberibacter americanus,” and “Ca. Liberibacter asiaticus” are associated with citrus greening disease, also referred as Huanglongbing (HLB) in different regions around the globe (Gottwald, 2010). “Ca. Liberibacter solanacearum” (=“Ca. Liberibacter psyllaurous”) is phytopathogenic to members of the Apiaceae and Solanaceae plant families. These four species all depend on psyllid vectors for transmission and as alternative hosts (Fagen et al., 2014b; Haapalainen, 2014). “Ca. Liberibacter europaeus” has also been associated with psyllids, but its role as a plant pathogen has not been demonstrated. To date, only Liberibacter crescens has been cultured in vitro, but it is not considered phytopathogenic and it is not vector-borne (Fagen et al., 2014a). L. crescens was first isolated as a bacterial endophyte from papaya and has not been re-isolated in nature. Non-psyllid hemipterans may also be able to pick up the bacteria during feeding as bacterial DNA has been found in mealybugs (Pitino et al., 2014), however, liberibacter transmission by other hemipterans is currently not clear.

Liberibacters have a small genome of ~1.2 Mb. Comparative genomics have shown a similar gene organization across the genus and evidence of horizontal gene transfers as prophages integrated into the genomes (Table 1) (Thompson et al., 2015). Similar to phytoplasmas, liberibacters lack biosynthesis genes for amino acids, sugars, and nitrogenated bases, which imply they obtain those metabolic products from their host (Thompson et al., 2015). Accordingly, many ABC transporters are encoded in liberibacter genomes (Lin et al., 2011; Mafra et al., 2013; Yan et al., 2013). Active importation of nutrients from phloem and insect vectors may lead to nutrient imbalances, partially explaining the foliar symptoms observed in liberibacter-infected plants (Rashed et al., 2013).

Potential pathogenicity mechanisms of liberibacters have recently been suggested based on comparative bioinformatics with other phloem-limited bacteria. Liberibacters encode the basic proteins for Sec-dependent translocation, similar to phytoplasmas (Lin et al., 2011; Yan et al., 2013). However, as liberibacters have two membranes of different composition in contrast to phytoplasmas, it is not known whether putative liberibacter Sec-transported proteins cross the outer membrane and interact with the plant or insect host. Recently, two unusual autotransporters were identified in the liberibacter genome (LasAI and LasAII) and these may serve as an alternative secretion system to the Sec-system (Table 2) (Hao et al., 2013). Evidence suggests that plant transcripts and metabolites related to salicylic acid (SA) production are altered during ‘Candidatus Liberibacter solanacerum’ infection (Casteel et al., 2012; Chin et al., 2014). SA is an important signaling molecule involved in plant defense to pathogens and insects (Glazebrook, 2005; Walling, 2009; Erb et al., 2012). Recently, a NahG-like salicylate hydroxylase gene was found in the liberibacter genome. NahG is predicted to cleave salicylates derived from SA (Lin et al., 2013) and may be used to modify the plant defense system. Although comparative bioinformatics has revealed many potential proteins used by liberibacter to alter plant and vector metabolism and vector–plants interactions, exact mechanisms for host colonization and transmission remain largely unknown.

The complete genome sequences for many strains of vector-borne bacteria have recently become available (Table 1). This has allowed scientists to study bacterial gene function within these systems without the need to culture the organism. Bioinformatics can be used to compare genome sequences with the annotated genomes of close relatives or analyze sequences using server-based algorithms to assign predicted functions to each coding region (Rutherford et al., 2000). Amino acid sequences can be further explored to identify conserved patterns and domains. In this way, proteins with low average similarity can be assigned to a predicted function (Yu et al., 2010; Caccia et al., 2013; Cortazar et al., 2015). Finally, functions of unknown proteins can even be predicted using dedicated algorithms that identify patterns associated with signal peptides, localization, cleavage sites, phosphorylation, and transmembrane domains (Yu et al., 2010).

A limitation of these various bioinformatics approaches is that all programs are trained using cultured organisms. For unculturable bacteria, many unique sequences with no homologs in cultured species exist, making comparisons and inferences difficult (Kube et al., 2008). Despite these limitations, bioinformatics have been used successfully to study gene function for many phytoplasma effectors. Bai et al. (2009) identified 56 Secreted Aster Yellows Proteins (SAPs) in the genome of ‘Candidatus Phytoplasma asteris’ strain Aster Yellows Witches’-broom (AY-WB). In this study, they utilized a pipeline to predict prokaryotic signal peptides recognized by Sec-translocases (SignalP v. 3.0) and then predicted transmembrane domains within this list to predict secretion (TMHMM v. 2.0; Bai et al., 2009). Finally, the list of 56 predicted effectors was examined for eukaryotic nuclear localization signals (predictNLS and pSORT) to select SAPs targeting plant nuclei for further investigation (Bai et al., 2009; Lu et al., 2014b).

After potential pathogenicity factors are identified, functional validation is required. For unculturable bacteria, transcription and translation of targets can only be evaluated within their hosts (plant or insect). This means RNA and protein isolations must be done from infected host tissue. By some estimates, only 0.1% of total RNA extracted from infected herbaceous hosts represents the phytoplasma RNA. Others report only 0.02% of the mRNA from the woody host was associated with the phytoplasma genome (Abba et al., 2014). However, high throughput sequencing technologies have expanded the possibilities for studying pathogens inside their hosts. Now RNAseq can be used to quantify the complete RNA population in a sample (Westermann et al., 2012). This technique has advantages over microarrays and qRT-PCR because it affords higher sensitivity for monitoring gene expression levels, independence from examining only known sequences, and wider detection ranges (Westermann et al., 2012). However, for vector-borne bacterial pathogens, RNAseq approaches have thus far had low levels of success.

RNAseq has been used to examine “Candidatus Phytoplasma mali” transcription in tobacco (Nicotiana occidentalis; Siewert et al., 2014). Prior to preparation for sequencing, total RNA was treated with a plant ribosomal depletion kit to enrich the samples for bacterial RNA. Only 0.003% of the total reads (17,046,418 reads averaging 115 b) were mapped against the protein coding regions of the predicted “Candidatus Phytoplasma mali” genome. Mapped reads corresponded to 132 genes out of the 497 predicted genes. In another RNAseq study, RNA was enriched for bacterial transcripts using a ribosomal depletion kit to remove plant cytoplasmic, mitochondrial, and chloroplast ribosomal RNA (Abba et al., 2014). Despite the enrichment and relatively deep sequencing, only 0.01% of the total reads (125,813,174 and 129,412,231, for each library) were mapped to the draft genome of the phytoplasma flavescence dore (Abba et al., 2014). In two slightly more successful studies, total RNA from psyllid vectors was used to detect transcripts from “Ca. Liberibacter solanacearum” (Ibanez et al., 2014; Yao et al., 2016). However, only 0.3% of the total (70,869,948) reads were mapped to the bacteria genome after ribosomal depletion (Ibanez et al., 2014). Transcriptomics offer a unique opportunity to overcome the many difficulties posed by these difficult pathosystems, but as evident in the above examples, many technical challenges remain.

The first approach that permitted gene function discovery for vector-borne bacterial plant pathogens was the use of transposon mutagenesis with spiroplasmas in the early 1990s (Fletcher et al., 1998; Mutaqin et al., 2011). In this approach, a transposon with a selective marker was integrated randomly into the chromosome of S. citri, and recombinant colonies were selected in media with antibiotics. When transformed colonies were tested in the host, the transposon was retained for a few days without antibiotic pressure. This technique was used to determine that disruption of a solute binding protein (gene sc76) reduced transmission in the leafhopper vector (Boutareaud et al., 2004). Since this first study, numerous research groups have generated collections of S. citri mutants using this technique (Foissac et al., 1997; Boutareaud et al., 2004; Mutaqin et al., 2011). Currently, X. fastidiosa and Spiroplasma spp. are the only vascular plant pathogens transmitted by hemipterans for which genetic transformation protocols and mutant libraries are currently available.

Genetic manipulation using surrogate culturable bacteria and heterologous gene expression in plants has been used to test gene function for other vector-borne bacteria (Jain et al., 2015; Renaudin et al., 2015). In a study using the flavescence dore phytoplasma, the surface protein, variable membrane protein A (VmpA), was expressed under the control of the S. citri tuf promoter in a recombinant S. citri (Table 2) (Renaudin et al., 2015). The tuf promoter was chosen because the tuf gene is expressed at high levels in most bacteria (Kim et al., 2009). In this system the leafhopper, Euscelidius variegatus, serves as a vector for both the phytoplasma and S. citri. Thus gain of function studies could be conducted with the recombinant S. citri in both hosts. In the case of phytopathogenic “Candidatus Liberibacter asiaticus,” a peroxidase protein (SC2_gp095) has been expressed in the cultivable L. crescens as a surrogate (Table 2). However, biological inferences from this system may be restricted by the lack of host infection of L. crescens after culturing.

An alternative method for studying gene function is to overexpress bacterial candidate proteins in the plant host. Model plants such as Arabidopsis thaliana and Nicotiana spp. are routinely used to evaluate gene function for plant pathogens. Once the candidate gene is selected, the coding sequence is cloned into a suitable expression vector and transgenic plants can be generated. Several authors have utilized plant heterologous expression systems to investigate the function of phytoplasma SAPs (MacLean et al., 2011; Lu et al., 2014a; Yang et al., 2015). In these studies transgenic A. thaliana expressing individual phytoplasma SAPs were screened for symptom development and plant abnormalities (MacLean et al., 2011; Lu et al., 2014a; Yang et al., 2015). After a relevant phenotype was identified, plant gene expression changes and plant proteins interacting with the phytoplasma proteins were examined (Table 2). A limitation of this approach is that only profound disturbances caused by a single bacterial gene can be identified. In addition, model plants may not serve as natural hosts for all vectored-borne bacteria and relevance of findings may be limited to an artificial system.