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. 2019 Dec 17;88(1):e00242-19. doi: 10.1128/IAI.00242-19

No Evidence of Apoptotic Response of the Potato Psyllid Bactericera cockerelli to “Candidatus Liberibacter solanacearum” at the Gut Interface

Xiao-Tian Tang a, Cecilia Tamborindeguy a,✉
Editor: De'Broski R Herbertb
PMCID: PMC6921667  PMID: 31611278

“Candidatus Liberibacter solanacearum” is a pathogen transmitted by the potato psyllid Bactericera cockerelli (Šulc) (Hemiptera: Triozidae) in a persistent manner. In this study, we investigated the molecular interaction between “Ca. Liberibacter solanacearum” and the potato psyllid at the gut interface. Specifically, we focused on the apoptotic response of potato psyllids to the infection by two “Ca. Liberibacter solanacearum” haplotypes, LsoA and LsoB.

KEYWORDS: Bactericera cockerelli, Liberibacter, cell death, immunity, psyllid, vector, zebra chip

ABSTRACT

“Candidatus Liberibacter solanacearum” is a pathogen transmitted by the potato psyllid Bactericera cockerelli (Šulc) (Hemiptera: Triozidae) in a persistent manner. In this study, we investigated the molecular interaction between “Ca. Liberibacter solanacearum” and the potato psyllid at the gut interface. Specifically, we focused on the apoptotic response of potato psyllids to the infection by two “Ca. Liberibacter solanacearum” haplotypes, LsoA and LsoB. To this end, we first quantified and localized “Ca. Liberibacter solanacearum” in the gut of adult psyllids. We then evaluated the existence of an apoptotic response in the insect gut using microscopy analyses to visualize the nuclei and the actin cytoskeleton of the gut cells and DNA fragmentation analyses by agarose gel electrophoresis. We also performed annexin V cell death assays to detect apoptosis. Finally, we annotated apoptosis-related genes from the potato psyllid transcriptome and evaluated their expression in response to “Ca. Liberibacter solanacearum” infection. The results showed no cellular markers of apoptosis despite the large amount of “Ca. Liberibacter solanacearum” present in the psyllid gut. In addition, only three genes potentially involved in apoptosis were regulated in the psyllid gut in response to “Ca. Liberibacter solanacearum”: the apoptosis-inducing factor AIF3 was downregulated in LsoA-infected psyllids, while the inhibitor of apoptosis IAPP5 was downregulated and IAP6 was upregulated in LsoB-infected psyllids. Overall, no evidence of apoptosis was observed in the gut of potato psyllid adults in response to either “Ca. Liberibacter solanacearum” haplotype. This study represents a first step toward understanding the interactions between “Ca. Liberibacter solanacearum” and the potato psyllid, which is crucial to developing approaches to disrupt their transmission.

INTRODUCTION

In the last 2 decades, several diseases associated with psyllid-borne bacterial pathogens have emerged worldwide, resulting in great economic losses. Some of these devastating diseases are caused by bacterial pathogens in the genus Liberibacter, including “Candidatus Liberibacter solanacearum,” “Candidatus Liberibacter americanus,” “Candidatus Liberibacter africanus,” and “Candidatus Liberibacter asiaticus” (1).

“Ca. Liberibacter solanacearum” is a phloem-limited, Gram-negative fastidious bacterium. It is the causative agent of potato zebra chip and other diseases in solanaceous crops in the United States, Mexico, Central America, and New Zealand (2). Presently, seven “Ca. Liberibacter solanacearum” haplotypes (LsoA, LsoB, LsoC, LsoD, LsoE, LsoF, and LsoU) have been identified in the world (3–7). In North America, the haplotypes LsoA and LsoB are transmitted by the potato psyllid (or tomato psyllid) Bactericera cockerelli (Šulc) (Hemiptera: Triozidae) (8). Similarly, “Ca. Liberibacter asiaticus,” another phloem-limited bacterium, causes the most devastating disease of citrus, huanglongbing. This bacterium is mainly transmitted by the Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae).

Both “Ca. Liberibacter solanacearum” and “Ca. Liberibacter asiaticus” are transmitted in a circulative and persistent manner (9–12). After being acquired from infected plants, these pathogens first colonize the psyllid gut. After replicating in the gut, these bacteria proceed to the hemolymph and infect other insect tissues, including the salivary glands, prior to their inoculation into the host plants during a subsequent feeding. Despite our understanding of their invasion route within the psyllid body, the mechanisms underlying the transmission of these two pathogens by the vectors remain largely unknown.

The gut, as the first organ that “Ca. Liberibacter solanacearum” and “Ca. Liberibacter asiaticus” encounter, provides an essential link for understanding Liberibacter transmission by psyllid vectors. Recent reports indicate that “Ca. Liberibacter asiaticus” induces apoptosis in the gut of D. citri adults, while no evidence of apoptosis was found in the nymphal guts (13, 14). Furthermore, “Ca. Liberibacter asiaticus” titer increases at a higher rate when the bacterium is acquired by nymphs rather than by adults (15). Therefore, the induction of apoptosis in the gut of adults may be a factor explaining the developmental differences of “Ca. Liberibacter asiaticus” acquisition by the vector. Interestingly, no evidence of apoptosis was found in the gut of adult carrot psyllids infected with LsoD (16). In contrast to “Ca. Liberibacter asiaticus,” “Ca. Liberibacter solanacearum” can be efficiently acquired during the nymphal and adult phases. Importantly, although the parameters for acquisition, transmission, and retention of “Ca. Liberibacter solanacearum” by potato psyllids have been preliminarily investigated, the interactions between the potato psyllid and “Ca. Liberibacter solanacearum” are not as well understood as those of the “Ca. Liberibacter asiaticus”-D. citri system.

Therefore, in this study, we investigated the molecular interaction between the potato psyllid and LsoA and LsoB. Specifically, we explored whether either of these two “Ca. Liberibacter solanacearum” haplotypes triggered an apoptotic response in the gut of the adult potato psyllid. We employed a four-step approach to this aim. First, we investigated whether differences of accumulation or localization of LsoA and LsoB in the gut of the potato psyllid were observed. Second, we evaluated the occurrence of markers of apoptosis using microscopy, annexin V cell death assays, and DNA fragmentation assays. Third, we annotated a set of apoptosis-related genes using the potato psyllid transcriptome. Fourth, we evaluated the expression of the identified apoptosis-related genes in the psyllid gut in response to the infection with each “Ca. Liberibacter solanacearum” haplotype. This study advances our understanding of the interactions between “Ca. Liberibacter solanacearum” and the potato psyllid. Our study may also contribute to developing new strategies to control diseases caused by different Liberibacter bacteria.

RESULTS

Quantification and immunolocalization of “Ca. Liberibacter solanacearum” in the gut of potato psyllids.

To characterize “Ca. Liberibacter solanacearum” accumulation in the gut, we first quantified “Ca. Liberibacter solanacearum” in pools of 50 guts of adult potato psyllids by quantitative real-time PCR (qPCR). The quantification results showed that there were approximately 1.0 × 107 to 3.0 × 107 genomes of LsoA or LsoB per pool, and there was no significant difference between them (P > 0.05) (Fig. 1A). “Ca. Liberibacter solanacearum” then was immunolocalized in the gut of LsoA- and LsoB-infected adults (Fig. 1B and C). In both cases, “Ca. Liberibacter solanacearum” distribution was widespread in the gut, and a high signal level was observed in the filter chamber or along the actin cytoskeleton of the gut cells (Fig. 1B and C). In contrast, no signal was detected in the guts of “Ca. Liberibacter solanacearum”-free psyllids under the same conditions (Fig. 1B).

FIG 1.

FIG 1

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Quantification and immunolocalization of “Ca. Liberibacter solanacearum” in the alimentary canal of potato psyllids. (A) Copies of LsoA (gray) and LsoB (black) in pools of 50 guts from LsoA- and LsoB-infected adult psyllids, respectively (n = 3). (B) Immunolocalization of “Ca. Liberibacter solanacearum” in the midgut of potato psyllids from the “Ca. Liberibacter solanacearum”-free and the LsoA- and LsoB-infected colonies. DAPI-counterstained nuclei are blue, actin is stained in green with phalloidin, and “Ca. Liberibacter solanacearum” signal is in red; “Ca. Liberibacter solanacearum” is frequently observed along the actin cytoskeleton. Bar, 20 μm. (C) Immunolocalization of LsoA and LsoB in the filter chamber of potato psyllid. FC, filter chamber. Bar, 20 μm. Lso, “Ca. Liberibacter solanacearum.”

Nuclear morphology and actin cytoskeleton architecture.

The nuclear architecture and actin cytoskeleton of cells from LsoA- and LsoB-infected adult psyllids were observed and compared to those from “Ca. Liberibacter solanacearum”-free individuals. Nuclei from both LsoA- and LsoB-infected gut cells appeared regularly dispersed in the cells and were of uniform round shape and size based on DAPI staining (Fig. 2A, blue). No differences were observed between the “Ca. Liberibacter solanacearum”-infected and “Ca. Liberibacter solanacearum”-free psyllids when comparing the nuclear morphology of the gut cells (Fig. 2A). In addition, the numbers of regular and irregular nuclei were counted and compared among the “Ca. Liberibacter solanacearum”-infected and “Ca. Liberibacter solanacearum”-free guts. There were no differences in percentage of cells with irregular nuclei among the guts of “Ca. Liberibacter solanacearum”-free and LsoA- and LsoB-infected adult psyllids (P > 0.05) (Fig. 2B).

FIG 2.

FIG 2

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Evaluation of the occurrence of apoptosis by microscopy, annexin V cell death assay, and DNA degradation assay. (A) Phalloidin and annexin V staining of “Ca. Liberibacter solanacearum”-free and LsoA- and LsoB-infected psyllid guts. (Top) DAPI-counterstained nuclei are blue and actin is stained in green with phalloidin. (Bottom) For annexin V staining, DAPI-counterstained nuclei are blue. Bar, 20 μm. (B) Percentage of normal nuclei in gut epithelial cells of “Ca. Liberibacter solanacearum”-free and LsoA- and LsoB-infected psyllids. Nuclei from six representative guts from each colony were assessed and counted. (C) Assessment of DNA integrity by agarose gel electrophoresis following extraction from guts of “Ca. Liberibacter solanacearum”-free (N) and LsoA (A)- and LsoB (B)-infected psyllids. M represents molecular weight markers in base pairs. The figure was spliced to remove unrelated samples.

The actin cytoskeleton in the gut of “Ca. Liberibacter solanacearum”-free and “Ca. Liberibacter solanacearum”-infected adults was stained using phalloidin (Fig. 2A, green). In general, the actin filaments appeared organized without disruption, and no differences were observed among the “Ca. Liberibacter solanacearum”-free and “Ca. Liberibacter solanacearum”-infected insects.

Annexin V cell death assay.

An annexin V cell death assay was performed to further determine whether “Ca. Liberibacter solanacearum” induced apoptosis in the cells of the gut of potato psyllids. The assay showed similar staining patterns in insects from the different colonies: nonspecific signal was observed in the membrane of the gut cells from “Ca. Liberibacter solanacearum”-free and LsoA- or LsoB-infected adult psyllids, indicating that these cells were not undergoing apoptosis (Fig. 2A).

DNA fragmentation assay.

The integrity of the gut DNA from the “Ca. Liberibacter solanacearum”-free and LsoA- and LsoB-infected psyllids was evaluated by agarose gel electrophoresis. No differences among the samples were observed (Fig. 2C). In particular, there was no detection of a DNA ladder, which is a distinctive biochemical feature of apoptosis.

Apoptosis-related genes in potato psyllid.

Putative apoptosis-inducing factors (AIF), inhibitors of apoptosis (IAP), and caspases were identified from the psyllid transcriptome data sets (17). In total, twelve apoptosis-related genes were identified and their sequences were verified, except for IAP6, the Dbruce homolog. The gene names and abbreviations used here are listed in Table 1.

TABLE 1.

Names of Bactericera cockerelli apoptosis-related genes used in this paper

Gene name Designation
TP53-regulated inhibitor of apoptosis protein 1 TRIAP1
Inhibitor of apoptosis protein 1 IAP1
Inhibitor of apoptosis protein 2 IAPP2
Inhibitor of apoptosis protein 5 IAP5
Baculoviral IAP repeat-containing protein 5 IAPP5
Baculoviral IAP repeat-containing protein 5.2-like IAPP5.2
Baculoviral IAP repeat-containing protein 6-like IAP6
Apoptosis-inducing factor 1 AIF1
Apoptosis-inducing factor 3 AIF3
Caspase 1 Caspase 1
Caspase 2 Caspase 2
Caspase 3 Caspase 3
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Three caspases were identified: two putative initiator caspases with a long prodomain (caspase 2 and caspase 3) and one putative effector caspase (caspase 1). A Bayesian inferred phylogenetic analysis of B. cockerelli, Acyrthosiphon pisum, Bemisia tabaci, Diaphorina citri, and Drosophila melanogaster caspases clustered these sequences into three distinct clades (Fig. 3). Caspase 2 is predicted to contain one CARD (caspase activation and recruitment domain). This protein clustered with phloem-feeding insect caspases containing CARD, with the exception of B. tabaci caspase 2 (LOC109030899). A. pisum was the only species encoding two CARD-containing caspases (LOC100569019 and LOC100571705). These proteins clustered with D. melanogaster initiator caspases Dronc, which contains CARD, and Dredd.

FIG 3.

FIG 3

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Phylogenetic relationships between caspase sequences of B. cockerelli, D. melanogaster, A. pisum, and B. tabaci. Amino acid alignments of caspase sequences were used to build a phylogenetic tree using a Bayesian inferred method with the C. elegans caspase CED-3 as an outgroup. The numbers at the nodes denote posterior probabilities. The red sequences with dots (●) indicate the sequences from B. cockerelli. The prefixes Bc, Ap, and Bt indicate B. cockerelli, A. pisum, and B. tabaci, respectively.

Caspase 3 is another predicted caspase with a long prodomain. It clustered with other long prodomain-containing caspases (Fig. 3). The prodomains of these caspases were serine and threonine (ST) rich. Two D. citri proteins encoded by the loci LOC10350169 and LOC103522950 clustered in this group. Both of these loci are probably incomplete, because they appear to lack the C-terminal region. Further, while the protein encoded by LOC103522950 does not have a long prodomain, it only encodes an 81-amino-acid-long protein with 100% identity to the protein encoded by LOC10350169.

One putative effector caspase was identified in B. cockerelli, caspase 1. This caspase clustered with short caspases (Fig. 3). A. pisum and D. citri each encode two putative effector caspases; however, both predicted loci of D. citri appear to be incomplete. This group of caspases clustered with D. melanogaster effector caspases Dcp-1 and DrICE.

Seven IAPs were identified in the potato psyllid transcriptome data set. We identified two deterin-like (IAPP5 and IAPP5.2), one DIAP1-like (IAP1), one DIAP2-like (IAPP2), one Dbruce-like (IAP6), one AAC11-like (IAP5), and one TP53-regulated inhibitor of apoptosis 1-like (TRIAP1) gene. Various numbers of IAPs were identified among the different phloem-feeding species (see Table S2 in the supplemental material). Of note, one homolog to AAC11 with an API5 domain was identified in each species, except in D. citri, in which three partial loci are predicted. No Dbruce homolog was identified in A. pisum, and several loci with partial similarity to these proteins, which probably represent different portions of the same gene, are predicted in the D. citri genome.

Two putative AIFs were identified in the potato psyllid transcriptome. One showed similarity to AIF3 from several insects, including D. citri and D. melanogaster uncharacterized protein Dmel_CG4199 (E value of 2e−164). The other AIF candidate was similar to AIF1 from several insects, including D. citri AIF1-like and D. melanogaster apoptosis-inducing factor. All phloem-feeding hemipterans encoded two putative AIFs, one with a Pyr-redox_2 and a AIF_c domain (AIF1-type) and one with a Rieske AIFL_N and a Pyr_redox_2 domain (AIF3). The only exception was D. citri, in which four loci are annotated as potential AIF1 (LOC103523312, LOC103516341, LOC108254404, and LOC103519937), three loci are annotated as AIF3 (LOC103523149, LOC113468611, and LOC103506257), and two loci are annotated as AIF3 pseudogenes (LOC103523148 and LOC103523147).

Expression of apoptosis-related genes.

The expression pattern of the identified apoptosis-related genes was evaluated in “Ca. Liberibacter solanacearum”-free and LsoA- and LsoB-infected gut samples. Of the twelve tested genes, only three were significantly regulated in response to “Ca. Liberibacter solanacearum” infection. Specifically, the inhibitor IAPP5 was significantly downregulated and IAP6 was upregulated in response to LsoB, while the inducer AIF3 was significantly downregulated in response to LsoA (Fig. 4). The other nine genes, in particular the three caspase genes, were not regulated in response to either LsoA or LsoB.

FIG 4.

FIG 4

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Regulation of apoptosis-related genes in the guts of “Ca. Liberibacter solanacearum”-free and LsoA- and LsoB-infected psyllids. (A) TRIAP1; (B) IAP1; (C) IAPP2; (D) IAP5; (E) IAPP5; (F) IAPP5.2; (G) IAP6; (H) AIF1; (I) AIF3; (J) caspase 1; (K) caspase 2; (L) caspase 3. N, “Ca. Liberibacter solanacearum” free; A, LsoA infected; B, LsoB infected. Data represent means ± standard deviations from three independent experiments. Different letters indicate statistical differences at a P value of <0.05 using one-way ANOVA with Tukey’s post hoc test.

DISCUSSION

Understanding the mechanisms underlying pathogen acquisition by insect vectors is critical for the development of effective strategies to control the diseases they cause. Recently, it was reported that apoptosis is induced in the gut of adult Asian citrus psyllids from “Ca. Liberibacter asiaticus”-infected colonies (13), but no evidence of apoptosis was found in the gut of the nymphs (13, 14) or in the gut of adult LsoD-infected carrot psyllids (16). Further, the “Ca. Liberibacter asiaticus” titer increases at a faster rate when the bacterium is acquired by nymphs than by adults (15). Therefore, the reduced apoptotic response of nymphs to “Ca. Liberibacter asiaticus” could be linked to the differences in acquisition between Asian citrus psyllid adults and nymphs. In this study, we examined the molecular interactions between “Ca. Liberibacter solanacearum” and the potato psyllid gut. Specifically, we first examined the accumulation of two “Ca. Liberibacter solanacearum” haplotypes, LsoA and LsoB, in the gut of adults, and second, we evaluated whether an apoptotic response was mounted in the gut.

“Ca. Liberibacter solanacearum” accumulation and colonization sites in the gut were examined by qPCR and immunolocalization, respectively. The results showed that both “Ca. Liberibacter solanacearum” haplotypes were abundant in the cytoplasm of gut cells. Strong “Ca. Liberibacter solanacearum”-derived signal was observed in the filter chamber and along the actin cytoskeleton of the gut cells. A similar pattern of accumulation was described for “Ca. Liberibacter asiaticus,” which was also located along the actin cytoskeleton of gut cells in the Asian citrus psyllid (18). In eukaryotic cells, the actin cytoskeleton mediates a variety of functions, including cell movement and intracellular trafficking (19). Because of its role, many intracellular bacterial pathogens use the host cell actin cytoskeleton to enter and move through the host cell (20). Some bacterial pathogens, such as Salmonella, secrete effector proteins that interact with and stimulate the rearrangement of the actin cytoskeleton, thereby promoting actin-based motility and cell-to-cell spread, facilitating further bacterial internalization and colonization in intestinal epithelium (21–24). We therefore hypothesize that actin cytoskeleton is involved in the colonization and translocation of Liberibacter bacteria in the psyllid gut.

We next evaluated whether apoptosis was induced in the potato psyllid gut. No evidence of apoptosis was obtained in the guts of “Ca. Liberibacter solanacearum”-infected adult psyllids based on nucleus morphology, actin cytoskeleton architecture, analysis of DNA fragmentation, or annexin V cell death assays. Two hypotheses for the reduced apoptotic response of potato psyllid adults in response to “Ca. Liberibacter solanacearum” infection can be proposed. First, the “Ca. Liberibacter solanacearum”-induced intracellular immune response did not reach or exceed the threshold to trigger an intracellular apoptotic immune reaction (25). Although “Ca. Liberibacter asiaticus” and “Ca. Liberibacter solanacearum” have similar genomes (26–29), their effects on the respective vectors are distinct (30, 31). Also, we speculate that other factors are involved in the interactions between psyllids and Liberibacter bacteria. For example, “Ca. Liberibacter asiaticus” and “Ca. Liberibacter solanacearum” infect different host plants, and the insects mount responses not just to the pathogen but also to the defense compounds from the infected plant (13). The second hypothesis is that “Ca. Liberibacter solanacearum” inhibits the insect immune response, which could lead to a high acquisition efficiency by potato psyllids. Indeed, potato psyllid adults efficiently transmit “Ca. Liberibacter solanacearum” even when the pathogen is acquired by adults (32). Therefore, future studies should compare the molecular interactions between psyllids and Liberibacter pathogens to decipher the differences in psyllid gut response to “Ca. Liberibacter solanacearum” and “Ca. Liberibacter asiaticus.” These differences could lead to the discovery of targets to disrupt the transmission of these and other bacterial pathogens by insects.

Because apoptosis has emerged as a potential mechanism involved in pathogen transmission, we identified putative apoptosis-related genes in the potato psyllid transcriptome and performed a comparative analysis with other phloem-feeding hemipterans that are also vectors of plant pathogens. We found that phloem-feeding hemipterans had a reduced set of caspase genes compared to that of D. melanogaster. We identified three putative caspases in B. cockerelli. Caspases 2 and 3 were characterized by long prodomains. Caspase 2 is predicted to encode a CARD that is found in initiator caspases, and it clustered with other CARD-containing caspases, including D. melanogaster Dronc, which is essential for apoptosis during multiple biological processes, including development (33). The caspase 3 prodomain was ST rich, which is a feature found only in insects, for example, in D. melanogaster Strica. No homolog to D. melanogaster Dredd was identified in the potato psyllid transcriptome or any of the other phloem-feeding insects analyzed. Dredd is under the control of the IMD pathway, activating innate immunity in response to Gram-negative bacteria (34). The absence of a Dredd homolog in the phloem-feeding insects evaluated fits the hypothesis that these insects lack a complete IMD pathway, as was determined based on genomic and transcriptomic sequence analysis of different phloem-feeding hemipterans, including the potato psyllid and the Asian citrus psyllid (17, 18, 35). Only one putative executioner caspase was identified, caspase 1, clustering with the D. melanogaster executioner caspases Dcp-1 and DrICE, which have overlapping functions (36).

On the other hand, more potential IAPs were identified in potato psyllids and in the other phloem-feeding hemipterans than in D. melanogaster. We identified a homolog of D. melanogaster DIAP1 that regulates cell death by binding and inactivating Dronc (37, 38) and can interact with DrICE and DCP-1 (39). We also identified a homolog of D. melanogaster DIAP2 in psyllids. DIAP2 exclusively regulates DrICE and does not bind other caspases (40). This protein is involved in the IMD pathway as a signal transducer downstream of the activation of Dredd instead of as a modulator of apoptosis (41). Similarly, we identified a homolog of Dbruce that inhibits apoptosis through ubiquitination of the IAP antagonist Reaper (42). We identified two homologs of Deterin, which controls cytokinesis (43). One AAC11 homolog was identified which prevents apoptosis after growth factor deprivation (44) by directly binding the caspase 2 CARD regulating cell death, autophagy, and aging (45). Finally, we identified a homolog of TP53-regulated inhibitor of apoptosis 1, a small conserved protein induced by TP53 under low levels of genotoxic stress, contributing to a reduction in cell death (46).

Two AIF proteins were identified in each phloem-feeding species. AIF proteins are mitochondrial oxidoreductase proteins critical for energy metabolism and induction of caspase-independent apoptosis.

Only three of these genes were regulated in response to “Ca. Liberibacter solanacearum” infection. AIF3 was downregulated in the gut of the LsoA-infected adult psyllids, while the deterin-like IAPP5 was downregulated and IAP6 was upregulated in the gut of LsoB-infected adult psyllids. None of the caspases were regulated in the gut of the potato psyllids in response to “Ca. Liberibacter solanacearum.” Caspases can be regulated in the gut of vectors in response to pathogens. For example, expression of Aedronc, Aedredd, and the Strica homolog caspase 16 increased in the gut of Aedes aegypti females from a refractory strain compared to that of the susceptible strain following a bloodmeal containing dengue-2 virus (47). Therefore, the gene expression results obtained here are in line with the previous evidence that “Ca. Liberibacter solanacearum” does not induce apoptosis in the gut of the adult potato psyllid.

Although the pathogen-vector systems of “Ca. Liberibacter asiaticus”-Asian citrus psyllid and “Ca. Liberibacter solanacearum”-potato psyllid are largely similar, the adaptive evolution of the insect vectors with the bacteria might have resulted in different infection strategies. Asian citrus psyllid might exploit the apoptotic response to limit the transmission of “Ca. Liberibacter asiaticus,” while this does not appear to occur in the potato or carrot psyllid in response to “Ca. Liberibacter solanacearum.” Therefore, the types of interaction between potato psyllid and “Ca. Liberibacter solanacearum” need to be evaluated. The information provided in this study in regard to “Ca. Liberibacter solanacearum” accumulation and the potato psyllid responses elicited by these bacteria may contribute to the development of alternative control strategies that could interfere with Liberibacter bacterial transmission.

MATERIALS AND METHODS

Insect colonies and gut tissue collection.

“Ca. Liberibacter solanacearum”-free and LsoA- and LsoB-infected psyllid colonies were maintained separately on tomato plants (moneymaker; Victory Seed Company, Molalla, OR) in insect-proof cages (24 by 13.5 by 13.5 cm; BioQuip, Compton, CA) at room temperature (24 ± 2°C) and under a photoperiod of 16 h light-8 h dark, as described in Yao et al. (48). Guts of “Ca. Liberibacter solanacearum”-free and LsoA- and LsoB-infected adult female psyllids (approximately 7 days old) were dissected under the dissecting microscope as described in Ibanez et al. (49).

Quantification of “Ca. Liberibacter solanacearum.”

To quantify “Ca. Liberibacter solanacearum” in the gut of LsoA- or LsoB-infected psyllids, DNA from pools of 50 guts was purified by following the protocol of the DNeasy blood and tissue kit (Qiagen, Hilden, Germany). Each pool represented one replicate and was used as an individual template for quantitative real-time PCR (qPCR) analyses. A total of three replicates were analyzed for each “Ca. Liberibacter solanacearum” haplotype. qPCR was performed using the SYBR green supermix kit (Bioline, Taunton, MA) according to the manufacturer’s instructions. Each reaction mixture included 25 ng of DNA, 250 nM each primer, and 1× SYBR green master mix. The volume was adjusted with nuclease-free water to 10 μl. The qPCR program was 95°C for 2 min, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. qPCR assays were performed using a QuantStudio 6 Flex real-time PCR system (Applied Biosystems, Foster City, CA). Reactions for all samples were performed in triplicates with a negative control in each run. The “Ca. Liberibacter solanacearum”-specific primers for the gene encoding a hypothetical protein (WP_013462289.1) (HP1Lso F, 5′-GGAAAAGCACAGTCAGTTTATG-3′; HP1Lso R, 5′-GGCAATTCGCAACTTAGACA-3′) were used for “Ca. Liberibacter solanacearum” quantification in psyllids. This gene is only found in “Ca. Liberibacter solanacearum,” and the specificity of the primers was verified using the Primer-BLAST tool from NCBI, searching against the nr database. Psyllid 28S rDNAF and 28S rDNAR primers (50) were used to amplify the 28S rRNA gene, which was used as an internal control. The average threshold cycle (CT) value from the three replicates was used to quantify the “Ca. Liberibacter solanacearum” levels. Data are reported as ΔCT = (CT of HP1Lso gene) − (CT of psyllid 28S gene). The “Ca. Liberibacter solanacearum” copy number in each sample was estimated by comparing the ΔCT value of the sample to a standard curve prepared by following the methods described in Levy et al. (51).

Immunolocalization of “Ca. Liberibacter solanacearum.”

To visualize “Ca. Liberibacter solanacearum” in the “Ca. Liberibacter solanacearum”-infected psyllid alimentary canal tissues, psyllid guts were first dissected in 1× phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO) from “Ca. Liberibacter solanacearum”-free and LsoA- and LsoB-infected adult psyllids and fixed in 4% paraformaldehyde for 30 min at room temperature. After fixation, the guts were incubated with Sudan Black B (SBB) (Sigma-Aldrich) for 20 min to quench autofluorescence, as described in Tang et al. (52). The guts then were permeabilized by incubating in 0.1% Triton X-100 (Calbiochem/EMD Chemicals, Gibbstown, NJ) for 30 min at room temperature. After washing three times with PBS containing 0.05% Tween 20 (PBST), blocking was performed for 1 h at room temperature with blocking buffer (PBST with 1% [wt/vol] bovine serum albumin). “Ca. Liberibacter solanacearum” immunolocalization was performed as described in Tang et al. (52) using a rabbit-derived polyclonal antibody (GenScript Corp., Piscataway, NJ) raised against the synthesized peptide “Ca. Liberibacter solanacearum” OMP-B VIRRELGFSEGDPIC. The guts were incubated with the antibody (diluted 1:500) overnight at 4°C. The guts then were washed three times with PBST and incubated with an Alexa Fluor 594-labeled goat anti-rabbit IgG secondary antibody (diluted 1:2,000; Invitrogen, Carlsbad, CA) for 1 h at room temperature. Guts were washed again three times with PBST and mounted with one drop of Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories Inc., Burlingame, CA) on a microscope slide. The slide was covered with a coverslip and sealed with nail polish. At least 20 guts per colony were examined using an AxioImager A1 microscope (Carl Zeiss Microimaging, Thornwood, NY) with the rhodamine filter (594 nm, red), and the images were collected and analyzed with AxioVision, release 4.8, software (Carl Zeiss).

Nuclear morphology, actin cytoskeleton architecture, and annexin V cell death assays.

To test whether “Ca. Liberibacter solanacearum” infection resulted in apoptosis in the potato psyllid gut, the nuclear morphology and the actin cytoskeleton architecture of the gut cells of adult potato psyllids from the “Ca. Liberibacter solanacearum”-free and the LsoA- and LsoB-infected colonies were observed. For that, the guts of adult insects from each colony were dissected, fixed, and incubated with SBB as previously described. The guts next were incubated with Alexa Fluor 488-labeled phalloidin (dilution, 1:200; Invitrogen) for 30 min. The guts were washed three times with PBST and mounted with one drop of Vectashield mounting medium with DAPI (Vector Laboratories Inc.).

Similarly, annexin staining was performed using the annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Abcam, Mountain View, CA) by following the manufacturer’s instructions. For that, after the guts were dissected in 1× PBS as described above, they were resuspended with 1× binding buffer and incubated with annexin V-FITC for 30 min in the dark. The guts then were fixed with 4% paraformaldehyde in 1× PBS for 15 min, washed with binding buffer three more times, mounted on microscope slides, covered with a coverslip, and sealed with nail polish.

At least 20 guts per colony and treatment were examined. All of the guts were examined using an AxioImager A1 microscope (Carl Zeiss Microimaging) with the FITC (488 nm, green) and rhodamine (594 nm, red) filters. The images were collected and analyzed with AxioVision, release 4.8, software (Carl Zeiss).

DNA fragmentation assay.

Apoptosis is characterized by DNA fragmentation, producing a characteristic DNA ladder. To test the integrity of the genomic DNA in the gut cells, the guts of “Ca. Liberibacter solanacearum”-free and LsoA- and LsoB-infected insects were dissected as previously described. A total of three replicates were analyzed for each colony. Genomic DNA was purified using the DNeasy blood and tissue kit (Qiagen), and subsequently the DNA samples were treated with RNase A (Invitrogen). Approximately 1 μg of DNA per sample was separately subjected to electrophoretic analysis. The DNA samples were run on 2% agarose gels containing ethidium bromide and visualized using UV light.

Identification of apoptosis-related genes.

Genes potentially involved in apoptosis were identified through BLAST searches of the psyllid transcriptome (17) using predicted genes of Drosophila melanogaster, Acyrthosiphon pisum, Bemisia tabaci, and D. citri as the query. The gene structure or domains of all apoptosis-related genes were identified with the Conserved Domain tool in NCBI.

To validate the bioinformatic predictions, primers for each candidate gene (except for IAP6, the homolog of Dbruce) were designed (see Table S1 in the supplemental material). RNA from a pool of 50 psyllid adults was purified using the RNeasy minikit (Qiagen). The total RNA then was reverse transcribed using the Verso cDNA synthesis kit (Thermo, Waltham, MA) and anchored oligo(dT) primers by following the manufacturer’s instructions. Candidate genes were amplified by PCR. The PCR conditions were 95°C for 2 min; 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 to 90 s (depending on amplicon size); and a final extension at 72°C for 5 min. Amplicons were visualized in a 1% agarose gel, and amplicons of the expected size were excised from the gel and purified using the PureLink quick gel extraction kit (Invitrogen). Each PCR fragment (150 ng) was cloned into the pGEM-T easy vector using the pGEM-T easy cloning kit (Promega, Madison, WI) and transformed into NovaBlue Singles competent cells (Novagen, Temecula, CA). For each construct, plasmid DNA from at least three colonies was purified using the PureLink quick plasmid miniprep kit (Invitrogen) and sequenced by Eton Bioscience, Inc. (San Diego, CA, USA). The obtained sequences were compared to the bioinformatics predictions.

For the phylogenetic analysis, potato psyllid caspase sequences were aligned with caspases from D. melanogaster, D. citri, A. pisum, and B. tabaci using ClustalW with the MEGA 5.2 software (53). The phylogenetic reconstruction was done by Bayesian inference using MrBayes 3.2 (54) with the Caenorhabditis elegans caspase CED-3 as an outgroup. Markov chain Monte Carlo runs were carried out for 2,000,000 generations, and the first 25% of sampled trees were discarded as burn-in. Tree information was visualized and edited using FigTree, v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree).

Expression of apoptosis-related genes.

The guts of “Ca. Liberibacter solanacearum”-free and LsoA- or LsoB-infected psyllids were dissected in 1× PBS with RNAlater (Ambion, Invitrogen) under the stereomicroscope. Guts from 200 adult psyllids of mixed sex and age were pooled. RNA purification from each pool was performed as described above, and genomic DNA was eliminated by DNase I treatment with Turbo DNase (Ambion, Invitrogen). cDNA synthesis from each pool was performed as described above. Each pool represented one replicate. A total of three replicates were analyzed for each colony. The expression of apoptosis-related genes in the “Ca. Liberibacter solanacearum”-free and “Ca. Liberibacter solanacearum”-infected guts was evaluated by qPCR using the SensiFAST SYBR Hi-ROX kit (Bioline) according to the manufacturer’s instructions as described above. The primers for qPCR are listed in Table S1. The relative expression of the candidate genes was estimated with the ΔΔCT method (55) using the two reference genes elongation factor-1a (GenBank accession no. KT185020) and ribosomal protein subunit 18 (GenBank accession no. KT279693), since they are the most stable genes for analyzing gene expression in the presence of “Ca. Liberibacter solanacearum” (56).

Data analysis.

All data were analyzed with JMP, version 12 (SAS Institute Inc., Cary, NC, USA). Quantification of LsoA and LsoB was compared with Student’s t tests. The percentage of normal nuclei and regulation of apoptosis-related genes was determined using one-way analysis of variance (ANOVA) with Tukey’s post hoc test.

Supplementary Material

Supplemental file 1
IAI.00242-19-s0001.pdf (210.5KB, pdf)

ACKNOWLEDGMENTS

We thank Freddy Ibanez and Julien Levy for comments. We thank Maria Azucena Mendoza Herrera for her help maintaining insect colonies and plants.

This work was supported by Texas A&M University and Texas A&M AgriLife Research (Controlling Exotic and Invasive Insect-Transmitted Pathogens) and Hatch project TEX0-1-9381, accession number 1015773. X.T. received the Herb Dean ‘40 Endowed Scholarship from the Department of Entomology at Texas A&M University.

We thank the Agriculture Women Excited to Share Opinions, Mentoring and Experiences (AWESOME) faculty group of the College of Agriculture and Life Sciences at Texas A&M University for assistance with editing the manuscript.

Footnotes

Supplemental material is available online only.

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Supplementary Materials

Supplemental file 1
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