Evaluation of a low-cost staining method for improved visualization of sweet potato whitefly (Bemisia tabaci) eggs on multiple crop plant species.

BACKGROUND: The sweet potato whitefly (Bemisia tabaci) is a globally important insect pest that damages crops through direct feeding and by transmitting viruses. Current B. tabaci management revolves around the use of insecticides, which are economically and environmentally costly. Host plant resistance is a sustainable option to reduce the impact of whiteflies, but progress in deploying resistance in crops has been slow. A major obstacle is the high cost and low throughput of screening plants for B. tabaci resistance. Oviposition rate is a popular metric for host plant resistance to B. tabaci because it does not require tracking insect development through the entire life cycle, but accurate quantification is still limited by difficulties in observing B. tabaci eggs, which are microscopic and translucent. The goal of our study was to improve quantification of B. tabaci eggs on several important crop species: cassava, cowpea, melon, sweet potato and tomato. RESULTS: We tested a selective staining process originally developed for leafhopper eggs: submerging the leaves in McBryde's stain (acetic acid, ethanol, 0.2% aqueous acid Fuchsin, water; 20:19:2:1) for three days, followed by clearing under heat and pressure for 15 min in clearing solution (LGW; lactic acid, glycerol, water; 17:20:23). With a less experienced individual counting the eggs, B. tabaci egg counts increased after staining across all five crops. With a more experienced counter, egg counts increased after staining on melons, tomatoes, and cowpeas. For all five crops, there was significantly greater agreement on egg counts across the two counting individuals after the staining process. The staining method worked particularly well on melon, where egg counts universally increased after staining for both counting individuals. CONCLUSIONS: Selective staining aids visualization of B. tabaci eggs across multiple crop plants, particularly species where leaf morphological features obscure eggs, such as melons and tomatoes. This method is broadly applicable to research questions requiring accurate quantification of B. tabaci eggs, including phenotyping for B. tabaci resistance.

A socially polymorphic Formica ant species exhibits a novel distribution of social supergene genotypes.

Most supergenes discovered so far are young, occurring in one species or a few closely related species. An ancient supergene in the ant genus Formica presents an unusual opportunity to compare supergene-associated phenotypes and the factors that influence the persistence of polymorphism in different species. We investigate the genetic architecture of social organization in Formica francoeuri, an ant species native to low- and mid-elevation semiarid regions of southern California, and describe an efficient technique for estimating mode of social organization using population genomic data. Using this technique, we show that F. francoeuri exhibits polymorphism in colony social organization and that the phenotypic polymorphism is strongly associated with genotypes within the Formica social supergene region. The distribution of supergene haplotypes in F. francoeuri differs from that of related species Formica selysi in that colonies with multiple queens contain almost exclusively workers that are heterozygous for alternative supergene haplotypes. Moreover, heterozygous workers exhibit allele-specific expression of the polygyne-associated haplotype at the candidate gene Knockout, which is thought to influence social organization. We also report geographic population structure and variation in worker size across a large fraction of the species range. Our results suggest that, although the Formica supergene is conserved within the genus, the mechanisms that maintain the supergene and its associated polymorphisms differ among species.

Uncovering Symbionts Across the Psyllid Tree of Life and the Discovery of a New Liberibacter Species, "Candidatus" Liberibacter capsica.

Sap-feeding insects in the order Hemiptera associate with obligate endosymbionts that are required for survival and facultative endosymbionts that can potentially modify resistance to stress, enemies, development, and reproduction. In the superfamily Psylloidea, the jumping plant lice (psyllids), less is known about the diversity and prevalence of their endosymbionts compared to other sap-feeding pests such as aphids (Aphididae). To address this knowledge gap, using 16S rRNA sequencing we identify symbionts across divergent psyllid host lineages from around the world. Taking advantage of a new comprehensive phylogenomic analyses of Psylloidea, we included psyllid samples from 44 species of 35 genera of five families, collected from 11 international locations for this study. Across psyllid lineages, a total of 91 OTUs were recovered, predominantly of the Enterobacteriaceae (68%). The diversity of endosymbionts harbored by each psyllid species was low with an average of approximately 3 OTUs. Two clades of endosymbionts (clade 1 and 2), belonging to Enterobacteriaceae, were identified that appear to be long term endosymbionts of the psyllid families Triozidae and Psyllidae, respectively. We also conducted high throughput metagenomic sequencing on three Ca. Liberibacter infected psyllid species (Russelliana capsici, Trichochermes walkeri, and Macrohomotoma gladiata), initially identified from 16S rRNA sequencing, to obtain more genomic information on these putative Liberibacter plant pathogens. The phylogenomic analyses from these data identified a new Ca. Liberibacter species, Candidatus Liberibacter capsica, that is a potential pathogen of solanaceous crops. This new species shares a distant ancestor with Ca. L. americanus, which occurs in the same range as R. capsici in South America. We also detected the first association between a psyllid specializing on woody hosts and the Liberibacter species Ca. L. psyllaurous, which is a globally distributed pathogen of herbaceous crop hosts in the Solanaceae. Finally, we detected a potential association between a psyllid pest of figs (M. gladiata) and a Ca. Liberibacter related to Ca. L. asiaticus, which causes severe disease in citrus. Our findings reveal a wider diversity of associations between facultative symbionts and psyllids than previously reported and suggest numerous avenues for future work to clarify novel associations of ecological, evolutionary, and pathogenic interest.

An organizing feature of bumble bee life history: worker emergence promotes queen reproduction and survival in young nests.

Bumble bee queens initiate nests solitarily and transition to living socially once they successfully rear their first cohort of offspring. Bumble bees are disproportionately important for early season pollination, and many populations are experiencing dramatic declines. In this system, the onset of the social stage is critical for nest survival, yet the mechanisms that facilitate this transition remain understudied. Further, the majority of conservation efforts target the social stage of the bumble bee life cycle and do not address the solitary founding stage. We experimentally manipulated the timing of worker emergence in young nests of bumble bee (Bombus impatiens) queens to determine whether and how queen fecundity and survival are impacted by the emergence of workers in the nest. We found that queens with workers added to the nest exhibit increased ovary activation, accelerated egg laying, elevated juvenile hormone (JH) titres and also lower mortality relative to solitary queens. We also show that JH is more strongly impacted by the social environment than associated with queen reproductive state, suggesting that this key regulator of insect reproduction has expanded its function in bumble bees to also influence social organization. We further demonstrate that these effects are independent of queen social history, suggesting that this underlying mechanism promoting queen fecundity is reversible and short lived. Synchronization between queen reproductive status and emergence of workers in the nest may ultimately increase the likelihood of early nesting success in social systems with solitary nest founding. Given that bumble bee workers regulate queen physiology as we have demonstrated, the timing of early worker emergence in the nest likely impacts queen fitness, colony developmental trajectories and ultimately nesting success. Collectively, our findings underline the importance of conservation interventions for bumble bees that support the early nesting period and facilitate the production and maintenance of workers in young nests.

Author Correction: New Ca. Liberibacter psyllaurous haplotype resurrected from a 49-year-old specimen of Solanum umbelliferum: a native host of the psyllid vector.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

New Ca. Liberibacter psyllaurous haplotype resurrected from a 49-year-old specimen of Solanum umbelliferum: a native host of the psyllid vector.

Over the last century, repeated emergence events within the Candidatus Liberibacter taxon have produced pathogens with devastating effects. Presently, our knowledge of Ca. Liberibacter diversity, host associations, and interactions with vectors is limited due to a focus on studying this taxon within crops. But to understand traits associated with pathogen emergence it is essential to study pathogen diversity in wild vegetation as well. Here, we explore historical native host plant associations and diversity of the cosmopolitan species, Ca. L. psyllaurous, also known as Ca. L. solanacearum, which is associated with psyllid yellows disease and zebra chip disease, especially in potato. We screened tissue from herbarium samples of three native solanaceous plants collected near potato-growing regions throughout Southern California over the last century. This screening revealed a new haplotype of Ca. L. psyllaurous (G), which, based on our sampling, has been present in the U.S. since at least 1970. Phylogenetic analysis of this new haplotype suggests that it may be closely related to a newly emerged North American haplotype (F) associated with zebra chip disease in potatoes. Our results demonstrate the value of herbarium sampling for discovering novel Ca. Liberibacter haplotypes not previously associated with disease in crops.

Addressing Research Needs in the Field of Plant Virus Ecology by Defining Knowledge Gaps and Developing Wild Dicot Study Systems.

Viruses are ubiquitous within all habitats that support cellular life and represent the most important emerging infectious diseases of plants. Despite this, it is only recently that we have begun to describe the ecological roles of plant viruses in unmanaged systems and the influence of ecosystem properties on virus evolution. We now know that wild plants frequently harbor infections by diverse virus species, but much remains to be learned about how viruses influence host traits and how hosts influence virus evolution and vector interactions. To identify knowledge gaps and suggest avenues for alleviating research deficits, we performed a quantitative synthesis of a representative sample of virus ecology literature, developed criteria for expanding the suite of pathosystems serving as models, and applied these criteria through a case study. We found significant gaps in the types of ecological systems studied, which merit more attention. In particular, there is a strong need for a greater diversity of logistically tractable, wild dicot perennial study systems suitable for experimental manipulations of infection status. Based on criteria developed from our quantitative synthesis, we evaluated three California native dicot perennials typically found in Mediterranean-climate plant communities as candidate models: Cucurbita foetidissima (buffalo gourd), Cucurbita palmata (coyote gourd), and Datura wrightii (sacred thorn-apple). We used Illumina sequencing and network analyses to characterize viromes and viral links among species, using samples taken from multiple individuals at two different reserves. We also compared our Illumina workflow with targeted RT-PCR detection assays of varying costs. To make this process accessible to ecologists looking to incorporate virology into existing studies, we describe our approach in detail and discuss advantages and challenges of different protocols. We also provide a bioinformatics workflow based on open-access tools with graphical user interfaces. Our study provides evidence that dicot perennials in xeric habitats support multiple, asymptomatic infections by viruses known to be pathogenic in related crop hosts. Quantifying the impacts of these interactions on plant performance and virus epidemiology in our logistically tractable host systems will provide fundamental information about plant virus ecology outside of crop environments.

All 17 S-locus F-box proteins of the S2 - and S3 -haplotypes of Petunia inflata are assembled into similar SCF complexes with a specific function in self-incompatibility.

The collaborative non-self-recognition model for S-RNase-based self-incompatibility predicts that multiple S-locus F-box proteins (SLFs) produced by pollen of a given S-haplotype collectively mediate ubiquitination and degradation of all non-self S-RNases, but not self S-RNases, in the pollen tube, thereby resulting in cross-compatible pollination but self-incompatible pollination. We had previously used pollen extracts containing GFP-fused S2 -SLF1 (SLF1 with an S2 -haplotype) of Petunia inflata for co-immunoprecipitation (Co-IP) and mass spectrometry (MS), and identified PiCUL1-P (a pollen-specific Cullin1), PiSSK1 (a pollen-specific Skp1-like protein) and PiRBX1 (a conventional Rbx1) as components of the SCF(S) (2-) (SLF) (1) complex. Using pollen extracts containing PiSSK1:FLAG:GFP for Co-IP/MS, we identified two additional SLFs (SLF4 and SLF13) that were assembled into SCF(SLF) complexes. As 17 SLF genes (SLF1 to SLF17) have been identified in S2 and S3 pollen, here we examined whether all 17 SLFs are assembled into similar complexes and, if so, whether these complexes are unique to SLFs. We modified the previous Co-IP/MS procedure, including the addition of style extracts from four different S-genotypes to pollen extracts containing PiSSK1:FLAG:GFP, to perform four separate experiments. The results taken together show that all 17 SLFs and an SLF-like protein, SLFLike1 (encoded by an S-locus-linked gene), co-immunoprecipitated with PiSSK1:FLAG:GFP. Moreover, of the 179 other F-box proteins predicted by S2 and S3 pollen transcriptomes, only a pair with 94.9% identity and another pair with 99.7% identity co-immunoprecipitated with PiSSK1:FLAG:GFP. These results suggest that SCF(SLF) complexes have evolved specifically to function in self-incompatibility.

Pollen S-locus F-box proteins of Petunia involved in S-RNase-based self-incompatibility are themselves subject to ubiquitin-mediated degradation.

Many flowering plants show self-incompatibility, an intra-specific reproductive barrier by which pistils reject self-pollen to prevent inbreeding and accept non-self pollen to promote out-crossing. In Petunia, the polymorphic S-locus determines self/non-self recognition. The locus contains a gene encoding an S-RNase, which controls pistil specificity, and multiple S-locus F-box (SLF) genes that collectively control pollen specificity. Each SLF is a component of an SCF (Skp1/Cullin/F-box) complex that is responsible for mediating degradation of non-self S-RNase(s), with which the SLF interacts, via the ubiquitin-26S proteasome pathway. A complete set of SLFs is required to detoxify all non-self S-RNases to allow cross-compatible pollination. Here, we show that SLF1 of Petunia inflata is itself subject to degradation via the ubiquitin-26S proteasome pathway, and identify an 18 amino acid sequence in the C-terminal region of S2 -SLF1 (SLF1 of S2 haplotype) that contains a degradation motif. Seven of the 18 amino acids are conserved among all 17 SLF proteins of S2 haplotype and S3 haplotype involved in pollen specificity, suggesting that all SLF proteins are probably subject to similar degradation. Deleting the 18 amino acid sequence from S2 -SLF1 stabilized the protein but abolished its function in self-incompatibility, suggesting that dynamic cycling of SLF proteins is an integral part of their function in self-incompatibility.

Insight into S-RNase-based self-incompatibility in Petunia: recent findings and future directions.

S-RNase-based self-incompatibility in Petunia is a self/non-self recognition system that allows the pistil to reject self-pollen to prevent inbreeding and to accept non-self pollen for outcrossing. Cloning of S-RNase in 1986 marked the beginning of nearly three decades of intensive research into the mechanism of this complex system. S-RNase was shown to be the sole female determinant in 1994, and the first male determinant, S-locus F-box protein1 (SLF1), was identified in 2004. It was discovered in 2010 that additional SLF proteins are involved in pollen specificity, and recently two S-haplotypes of Petunia inflata were found to possess 17 SLF genes based on pollen transcriptome analysis, further increasing the complexity of the system. Here, we first summarize the current understanding of how the interplay between SLF proteins and S-RNase in the pollen tube allows cross-compatible pollination, but results in self-incompatible pollination. We then discuss some of the aspects that are not yet elucidated, including uptake of S-RNase into the pollen tube, nature, and assembly of SLF-containing complexes, the biochemical basis for differential interactions between SLF proteins and S-RNase, and fate of non-self S-RNases in the pollen tube.

Identification of the self-incompatibility locus F-box protein-containing complex in Petunia inflata.

The polymorphic S-locus regulating self-incompatibility (SI) in Petunia contains the S-RNase gene and a number of S-locus F-box (SLF) genes. While penetrating the style through the stigma, a pollen tube takes up all S-RNases, but only self S-RNase inhibits pollen tube growth. Recent evidence suggests that SLFs produced by pollen collectively interact with and detoxify non-self S-RNases, but none can interact with self S-RNase. An SLF may be the F-box protein component of an SCF complex (containing Cullin1, Skp1 and Rbx1), which mediates ubiquitination of protein substrates for degradation by the 26S proteasome. However, the precise nature of the complex is unknown. We used pollen extracts of a transgenic plant over-expressing GFP-fused S2-SLF1 (SLF1 of S 2-haplotype) for co-immunoprecipitation (Co-IP) followed by mass spectrometry (MS). We identified PiCUL1-P (a pollen-specific Cullin1), PiSSK1 (a pollen-specific Skp1-like protein) and PiRBX1 (an Rbx1). To validate the results, we raised transgenic plants over-expressing PiSSK1:FLAG:GFP and used pollen extracts for Co-IP-MS. The results confirmed the presence of PiCUL1-P and PiRBX1 in the complex and identified two different SLFs as the F-box protein component. Thus, all but Rbx1 of the complex may have evolved in SI, and all SLFs may be the F-box component of similar complexes.

Self-incompatibility in Petunia inflata: the relationship between a self-incompatibility locus F-box protein and its non-self S-RNases.

The highly polymorphic S (for self-incompatibility) locus regulates self-incompatibility in Petunia inflata; the S-RNase regulates pistil specificity, and multiple S-locus F-box (SLF) genes regulate pollen specificity. The collaborative non-self recognition model predicts that, for any S-haplotype, an unknown number of SLFs collectively recognize all non-self S-RNases to mediate their ubiquitination and degradation. Using a gain-of-function assay, we examined the relationships between S2-SLF1 (for S2-allelic product of Type-1 SLF) and four S-RNases. The results suggest that S2-SLF1 interacts with S7- and S13-RNases, and the previously identified S1- and S3-RNases, but not with S5- or S11-RNase. An artificial microRNA expressed by the S2-SLF1 promoter, but not by the vegetative cell-specific promoter, Late Anther Tomato 52, suppressed expression of S2-SLF1 in S2 pollen, suggesting that SLF1 is specific to the generative cell. The S2 pollen with S2-SLF1 suppressed was compatible with S3-, S5-, S7-, S11-, and S13-carrying pistils, confirming that other SLF proteins are responsible for detoxifying S5- and S11-RNases and suggesting that S2-SLF1 is not the only SLF in S2 pollen that interacts with S3-, S7-, and S13-RNases. Petunia may have evolved at least two types of SLF proteins to detoxify any non-self S-RNase to minimize the deleterious effects of mutation in any SLF.

Identification of quantitative trait loci associated with germination using chromosome segment substitution lines of rice (Oryza sativa L.).

Rapid and uniform seed germination under diverse environmental conditions is a desirable characteristic for most crop plants, such as rice, wheat, and maize. However, the genetic base of the variations in the rate of germination is not well understood. In this study, quantitative trait loci (QTL) for germination rate were mapped with a set of 143 chromosome segment substitution lines (CSSL) each contains a small genomic fragment from a japonica variety Nipponbare in the uniform genetic background of an indica variety Zhenshan97. Nine CSSL showed significantly lower germination rate than that in Zhenshan97. Four germination-related QTL were identified located on chromosomes 2, 5, 6 and 10, at which all japonica alleles decreased germination rate. By using the CSSL-derived F2 population, a major QTL (qGR2) on chromosome 2 was confirmed, and delimited to a 10.4 kb interval containing three putative candidate genes, of which OsMADS29 was only expressed preferentially in the seed. These results would facilitate cloning of the major gene that affects germination rate, and provide an insight into the genetic basis of germination.

S-RNase-based self-incompatibility in Petunia inflata.

BACKGROUND: For the Solanaceae-type self-incompatibility, also possessed by Rosaceae and Plantaginaceae, the specificity of self/non-self interactions between pollen and pistil is controlled by two polymorphic genes at the S-locus: the S-locus F-box gene (SLF or SFB) controls pollen specificity and the S-RNase gene controls pistil specificity. SCOPE: This review focuses on the work from the authors' laboratory using Petunia inflata (Solanaceae) as a model. Here, recent results on the identification and functional studies of S-RNase and SLF are summarized and a protein-degradation model is proposed to explain the biochemical mechanism for specific rejection of self-pollen tubes by the pistil. CONCLUSIONS: The protein-degradation model invokes specific degradation of non-self S-RNases in the pollen tube mediated by an SLF, and can explain compatible versus incompatible pollination and the phenomenon of competitive interaction, where SI breaks down in pollen carrying two different S-alleles. In Solanaceae, Plantaginaceae and subfamily Maloideae of Rosaceae, there also exist multiple S-locus-linked SLF/SFB-like genes that potentially function as the pollen S-gene. To date, only three such genes, all in P. inflata, have been examined, and they do not function as the pollen S-gene in the S-genotype backgrounds tested. Interestingly, subfamily Prunoideae of Rosaceae appears to possess only a single SLF/SFB gene, and competitive interaction, observed in Solanaceae, Plantaginaceae and subfamily Maloideae, has not been observed. Thus, although the cytotoxic function of S-RNase is an integral part of SI in Solanaceae, Plantaginaceae and Rosaceae, the function of SLF/SFB may have diverged. This highlights the complexity of the S-RNase-based SI mechanism. The review concludes by discussing some key experiments that will further advance our understanding of this self/non-self discrimination mechanism.

The amino terminal F-box domain of Petunia inflata S-locus F-box protein is involved in the S-RNase-based self-incompatibility mechanism.

BACKGROUND AND AIMS: Pistils of flowering plants possessing self-incompatibility (SI) can distinguish between self and non-self pollen, and only allow non-self pollen to effect fertilization. For Petunia inflata, the S-RNase gene encodes pistil specificity and multiple S-locus F-box (SLF) genes encode pollen specificity. Each SLF produced in pollen interacts with a subset of non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome. RATIONALE: S-locus F-box has been proposed to function as a component of the conventional SCF (SKP1-CULLIN-F-box protein) complex, based on the finding that two SKP1-like proteins, AhSSK1 (Antirrhinum hispanicum SLF-interacting SKP1-like1) and PhSSK1 (Petunia hybrida SSK1), interact with the F-box domain of some allelic variants of SLF. However, we previously showed that PiSLF (P. inflata SLF) did not interact with any SKP1 of P. inflata or Arabidopsis thaliana, but instead interacted with a RING-finger protein, PiSBP1 (P. inflata S-RNase-Binding Protein1), which may also play the role of SKP1. Thus, the biochemical nature of the SLF-containing complex is as yet unclear. PRINCIPAL RESULTS: To examine whether the F-box domain of PiSLF is required for SI function, we expressed a truncated PiSLF(2) (S(2) allelic variant) without this domain in S(2)S(3) plants and showed that, unlike the full-length PiSLF(2), it did not cause breakdown of SI in S(3) pollen. We identified PiSSK1 (P. inflata SSK1) and found that it did not interact with PiSLF(1), PiSLF(2) or PiSLF(3). CONCLUSIONS: The finding that the truncated PiSLF(2) did not cause breakdown of SI in S(3) transgenic pollen suggests that the F-box domain of PiSLF(2) is required for mediating degradation of S(3)-RNase, a non-self S-RNase, in S(3) pollen, and thus is required for SI function. The finding that PiSSK1 did not interact with three allelic variants of PiSLF is consistent with our previous finding that PiSLF might not be in a conventional SCF complex.