Structural insights into IL-6 signaling inhibition by therapeutic antibodies.

Antibody inhibitors of the interleukin-6 (IL-6) signaling pathway, such as tocilizumab and sarilumab, have been used to treat rheumatoid arthritis, chimeric antigen receptor T cell-induced cytokine storm, and severe COVID-19 pneumonia. Here, we solve the cryogenic electron microscopy structures of sarilumab and tocilizumab in complex with IL-6R to resolutions of 3.2 and 3.3 Å, respectively. These structures reveal that both tocilizumab and sarilumab bind to the D3 domain of IL-6R. The binding surfaces of the two antibodies largely overlap, but the detailed interactions are different. Functional studies of various mutants show results consistent with our structural analysis of the antibodies and IL-6R interactions. Structural comparisons with the IL-6/IL-6R/gp130 complex indicate that sarilumab and tocilizumab probably inhibit IL-6/IL-6R signaling by competing for the IL-6 binding site. In summary, this work reveals the antibody-blocking mechanism of the IL-6 signaling pathway and paves the way for future antibody discovery.

The essential role of clathrin-mediated endocytosis and early endosomes in the trafficking of begomoviruses through the primary salivary glands of their whitefly vectors.

IMPORTANCE: Many plant viruses are transmitted by insect vectors in a circulative manner. For efficient transmission, the entry of the virus from vector hemolymph into the primary salivary gland (PSG) is a step of paramount importance. Yet, vector components mediating virus entry into PSG remain barely characterized. Here, we demonstrate the role of clathrin-mediated endocytosis and early endosomes in begomovirus entry into whitefly PSG. Our findings unravel the key components involved in begomovirus transport within the whitefly body and transmission by their whitefly vectors and provide novel clues for blocking begomovirus transmission.

RT-RPA-PfAgo System: A Rapid, Sensitive, and Specific Multiplex Detection Method for Rice-Infecting Viruses.

The advancement in CRISPR-Cas biosensors has transmuted the detection of plant viruses owing to their rapid and higher sensitivity. However, false positives and restricted multiplexing capabilities are still the challenges faced by this technology, demanding the exploration of novel methodologies. In this study, a novel detection system was developed by integrating reverse transcriptome (RT) techniques with recombinase polymerase isothermal amplification (RPA) and Pyrococcus furiosus Argonaute (PfAgo). The RT-RPA-PfAgo system enabled the simultaneous detection of rice ragged stunt virus (RRSV), rice grassy stunt virus (RGSV), and rice black streaked dwarf virus (RBSDV). Identifying targets via guide DNA without being hindered by protospacer adjacent motif sequences is the inherent merit of PfAgo, with the additional advantage of it being simple, cost-effective, and exceptionally sensitive, with detection limits between 3.13 and 5.13 copies/µL, in addition to it effectively differentiating between the three distinct viruses. The field evaluations were also in accordance with RT-PCR methods. The RT-RPA-PfAgo system proved to be a robust, versatile, highly specific, and sensitive method with great potential for practicality in future plant virus diagnostics.

Diploid mycelia of Ustilago esculenta fails to maintain sustainable proliferation in host plant.

Smut fungi display a uniform life cycle including two phases: a saprophytic phase in vitro and a parasitic phase in host plants. Several apathogenic smut fungi are found, lacking suitable hosts in their habitat. Interestingly, MT-type Ustilago esculenta was found to maintain a parasitic life, lacking the saprophytic phase. Its long period of asexual proliferation in plant tissue results in severe defects in certain functions. In this study, the growth dynamics of U. esculenta in plant tissues were carefully observed. The mycelia of T- and MT-type U. esculenta exhibit rapid growth after karyogamy and aggregate between cells. While T-type U. esculenta successfully forms teliospores after aggregation, the aggregated mycelia of MT-type U. esculenta gradually disappeared after a short period of massive proliferation. It may be resulted by the lack of nutrition such as glucose and sucrose. After overwintering, infected Zizania latifolia plants no longer contained diploid mycelia resulting from karyogamy. This indicated that diploid mycelia failed to survive in plant tissues. It seems that diploid mycelium only serves to generate teliospores. Notably, MT-type U. esculenta keeps the normal function of karyogamy, though it is not necessary for its asexual life in plant tissue. Further investigations are required to uncover the underlying mechanism, which would improve our understanding of the life cycle of smut fungi and help the breeding of Z. latifolia.

The Novel Effector Ue943 Is Essential for Host Plant Colonization by Ustilago esculenta.

The smut fungus Ustilago esculenta obligately parasitizes Zizania latifolia and induces smut galls at the stem tips of host plants. Previous research identified a putative secreted protein, Ue943, which is required for the biotrophic phase of U. esculenta but not for the saprophytic phase. Here, we studied the role of Ue943 during the infection process. Conserved homologs of Ue943 were found in smut fungi. Ue943 can be secreted by U. esculenta and localized to the biotrophic interface between fungi and plants. It is required at the early stage of colonization. The Ue943 deletion mutant caused reactive oxygen species (ROS) production and callose deposition in the host plant at 1 and 5 days post inoculation, which led to failed colonization. The virulence deficiency was restored by overexpressing gene Ue943 or Ue943:GFP. Transcriptome analysis further showed a series of changes in plant hormones following ROS production when the host plant was exposed to ΔUe943. We hypothesize that Ue943 might be responsible for ROS suppression or avoidance of recognition by the plant immune system. The mechanism underlying Ue943 requires further study to provide more insights into the virulence of smut fungi.

Construction a new nomogram prognostic model for predicting overall survival after radical resection of esophageal squamous cancer.

Background: Esophageal cancer is one of the deadliest malignancies in the world, and 5-year overall survival (OS) of esophageal cancer ranges from 12% to 20%. Surgical resection remains the principal treatment. The American Joint Commission on Cancer (AJCC) TNM (tumor, node, and metastasis) staging system is a key guideline for prognosis and treatment decisions, but it cannot fully predict outcomes. Therefore, targeting the molecular and biological features of each patient's tumor, and identifying key prognostic biomarkers as effective survival predictors and therapeutic targets are highly important to clinicians and patients. Methods: In this study, three different methods, including Univariate Cox regression, Lasso regression, and Randomforest regression were used to screen the independent factors affecting the prognosis of esophageal squamous cell carcinoma and construct a nomogram prognostic model. The accuracy of the model was verified by comparing with TNM staging system and the reliability of the model was verified by internal cross validation. Results: Preoperative neutrophil lymphocyte ratio(preNLR), N-stage, p53 level and tumor diameter were selected to construct the new prognostic model. Patients with higher preNLR level, higher N-stage, lower p53 level and larger tumor diameter had worse OS. The results of C-index, Decision Curve Analysis (DCA), and integrated discrimination improvement (IDI) showed that the new prognostic model has a better prediction than the TNM staging system. Conclusion: The accuracy and reliability of the nomogram prognostic model were higher than that of TNM staging system. It can effectively predict individual OS and provide theoretical basis for clinical decision making.

An Endoglucanase Secreted by Ustilago esculenta Promotes Fungal Proliferation.

Ustilago esculenta is a fungus of two morphological forms, among the filamentous dikaryon that can induce the plant stem to expand to form fleshy stem. In order to establish biotrophy with Zizania latifolia which belongs to the tribe Oryzeae (Poaceae), U. esculenta firstly needs to secrete a bunch of effectors, among them being cell wall degrading enzymes (CWDEs). We have isolated a gene, UeEgl1, which was differentially expressed in MT-type and T-type U. esculenta at an early stage of infection, and specifically induced in the filamentous growth of the T-type. Bioinformatics analysis and enzyme activity assay indicated that UeEgl1 functions outside the cell as a ß-1,4-endoglucanase with a conserved domain of the glycosyl hydrolase family 45 (GH45) which targets the main component of the plant cell wall ß-1,4 linked glycosidic bonds. The phenotype analysis of UeEgl1 deletion mutants and UeEgl1 over-expression transformants showed that UeEgl1 had no significant effect on the budding, cell fusion, and filamentous growth of U. esculenta in vitro. Further study found that over-expression of UeEgl1 promoted the proliferation of mycelia inside Z. latifolia, and raised plant defense responses. The above results show that the UeEgl1 gene may play an important role in the early stage of infection through the decomposition of the plant cell wall.

Transcriptome Comparison between Two Strains of Ustilago esculenta during the Mating.

Ustilago esculenta is a smut fungus that obligately infects Zizania latifolia and stimulates tissue swelling to form galls. Unlike T-type, MT-type U. esculenta can only proliferate within plant tissues and infect the offspring of their host. Production of telispores, haploid life, and plant cuticle penetration are not essential for it, which may lead to the degeneration in these processes. Transcriptome changes during the mating of T- and MT-type U. esculenta were studied. The functions of several secreted proteins were further confirmed by knock-out mutants. Our results showed that MT-type U. esculenta can receive environmental signals in mating and circumstance sensing as T-type does. However, MT-type U. esculenta takes a longer time for conjunction tube formation and cytoplasmic fusion. A large number of genes encoding secreted proteins are enriched in the purple co-expression module. They are significantly up-regulated in the late stage of mating in T-type U. esculenta, indicating their relationship with infecting. The knock-out of g6161 (xylanase) resulted in an attenuated symptom. The knock-out of g943 or g4344 (function unidentified) completely blocked the infection at an early stage. This study provides a comprehensive comparison between T- and MT-type during mating and identifies two candidate effectors for further study.

Functional Properties of the MAP Kinase UeKpp2 in Ustilago esculenta.

Ustilago esculenta undergoes an endophytic life cycle in Zizania latifolia. It induces the stem of its host to swell, forming the edible galls called jiaobai in China, which are the second most commonly cultivated aquatic vegetable in China. Z. latifolia raised for jiaobai can only reproduce asexually because the U. esculenta infection completely inhibits flowering. The infection and proliferation in the host plants during the formation of edible gall differ from those of conventional pathogens. Previous studies have shown a close relationship between mitogen-activated protein kinase (MAPK) and fungal pathogenesis. In this study, we explored the functional properties of the MAPK UeKpp2. Cross-species complementation assays were carried out, which indicated a functional complementation between the UeKpp2 of U. esculenta and the Kpp2 of Ustilago maydis. Next, UeKpp2 mutants of the UeT14 and the UeT55 sporidia background were generated; these showed an aberrant morphology of budding cells, and attenuated mating and filamentous growth in vitro, in the context of normal pathogenicity. Interestingly, we identified another protein kinase, UeUkc1, which acted downstream of UeKpp2 and may participate in the regulation of cell shape. We also found a defect of filamentous growth in UeKpp2 mutants that was not related to a defect of the induction of mating-type genes but was directly related to a defect in UeRbf1 induction. Overall, our results indicate an important role for UeKpp2 in U. esculenta that is slightly different from those reported for other smut fungi.

Smut fungal strategies for the successful infection.

The smut fungi include a large number of plant pathogens that establish obligate biotrophic relationships with their host. Throughout the whole life inside plant tissue, smut fungi keep plant cells alive and acquire nutrients via biotrophic interfaces. This mini-review mainly summarizes the interactions between smut fungi and their host plants during the infection process. Despite various strategies recruited by plants to defense invading pathogens, smut fungi successfully evolved an arsenal for colonization. Mating of two compatible haploids gives rise to parasitic mycelium, which can sense plant surface cues such as fatty acids and hydrophobic surface, and induce the formation of appressoria for surface penetration. Plants can recognize fungal invading and activate defense response, including callose and lignin deposition, programmed cell death, and SA signaling pathway. To suppress plant immunity and alter the metabolic pathway of host plants, a cocktail of effectors is secreted by smut fungi depending on the plant organ and cell type that is infected.

Cloning and disruption of the UeArginase in Ustilago esculenta: evidence for a role of arginine in its dimorphic transition.

BACKGROUND: Ustilago esculenta, a typical dimorphic fungus could infect Zizania latifolia and induce host stem swollen to form an edible vegetable called Jiaobai in China. The strains differentiation especially in the mating ability and pathogenicity is closely related to different phenotypes of Jiaobai formed in the fields. Dimorphic switching, a tightly regulated processes, is essential for the pathogenetic development of dimorphic fungi. In responses to environment cues, dimorphic switching can be activated through two conserved cell signaling pathways-PKA and MAPK pathways. Previous study indicated that exogenous arginine could induce hyphal formation in several dimorphic fungi through hydrolysis by arginase, but inhibit the dimorphic transition of U. esculenta. We conducted this study to reveal the function of arginine on dimorphic transition of U. esculenta. RESULTS: In this study, we found that arginine, but not its anabolites, could slow down the dimorphic transition of U. esculenta proportionally to the concentration of arginine. Besides, UeArginase, predicated coding arginase in U. esculenta was cloned and characterized. UeArginase mutants could actually increase the content of endogenous arginine, and slow down the dimorphic transition on either nutritious rich or poor medium. Either adding exogenous arginine or UeArginase deletion lead to down regulated expressions of UePkaC, UePrf1, mfa1.2, mfa2.1, pra1 and pra2, along with an increased content of arginine during mating process. CONCLUSION: Results of this study indicated a direct role of arginine itself on the inhibition of dimorphic transition of U. esculenta, independent of its hydrolysis by UeArginase.

Publisher Correction: Insight into the microbial world of Bemisia tabaci cryptic species complex and its relationships with its host.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Insight into the microbial world of Bemisia tabaci cryptic species complex and its relationships with its host.

The 37 currently recognized Bemisia tabaci cryptic species are economically important species and contain both primary and secondary endosymbionts, but their diversity has never been mapped systematically across the group. To achieve this, PacBio sequencing of full-length bacterial 16S rRNA gene amplicons was carried out on 21 globally collected species in the B. tabaci complex, and two samples from B. afer were used here as outgroups. The microbial diversity was first explored across the major lineages of the whole group and 15 new putative bacterial sequences were observed. Extensive comparison of our results with previous endosymbiont diversity surveys which used PCR or multiplex 454 pyrosequencing platforms showed that the bacterial diversity was underestimated. To validate these new putative bacteria, one of them (Halomonas) was first confirmed to be present in MED B. tabaci using Hiseq2500 and FISH technologies. These results confirmed PacBio is a reliable and informative venue to reveal the bacterial diversity of insects. In addition, many new secondary endosymbiotic strains of Rickettsia and Arsenophonus were found, increasing the known diversity in these groups. For the previously described primary endosymbionts, one Portiera Operational Taxonomic Units (OTU) was shared by all B. tabaci species. The congruence of the B. tabaci-host and Portiera phylogenetic trees provides strong support for the hypothesis that primary endosymbionts co-speciated with their hosts. Likewise, a comparison of bacterial alpha diversities, Principal Coordinate Analysis, indistinct endosymbiotic communities harbored by different species and the co-divergence analyses suggest a lack of association between overall microbial diversity with cryptic species, further indicate that the secondary endosymbiont-mediated speciation is unlikely to have occurred in the B. tabaci species group.

Mating-type loci of Ustilago esculenta are essential for mating and development.

Ustilago esculenta is closely related to the smut fungus Ustilago maydis and, in an endophytic-like life in the plant Zizania latifolia, only infects host stems and causes swollen stems to form edible galls called Jiaobai in China. In order to study its different modes of invasion and sites of symptom development from other smut fungi at the molecular level, we first characterized the a and b mating-type loci of U. esculenta. The a loci contained three a mating-type alleles, encoding two pheromones and one pheromone receptor per allele. The pheromone/receptor system controlled the conjugation formation, the initial step of mating, in which each pheromone was specific for recognition by only one mating partner. In addition, there are at least three b alleles identified in U. esculenta, encoding two subunits of heterodimeric homeodomain transcription factors bE and bW, responsible for hyphal growth and invasiveness. Hyphal formation, elongation and invasion after mating of two compatible partners occurred, only when a heterodimer complex was formed by the bE and bW proteins derived from different alleles. We also demonstrated that even with only one paired pheromone-pheromone receptor, the active b locus heterodimer triggered hyphal growth and infection.

Intracellular trafficking of begomoviruses in the midgut cells of their insect vector.

Begomoviruses are exclusively transmitted by whiteflies in a persistent circulative manner and cause considerable economic losses to crop production worldwide. Previous studies have shown that begomoviruses accumulate in vesicle-like structures in whitefly midgut cells and that clathrin-mediated endocytosis is responsible for their internalization. However, the process by which begomoviruses are trafficked within whitefly midgut cells remains largely unknown. In this study, we investigated the roles of vesicle trafficking in the transport of Tomato yellow leaf curl virus (TYLCV), a begomovirus that has spread to over 50 countries and caused extensive damage to a range of important crops, within midgut cells of whitefly (Bemisia tabaci). By disrupting vesicle trafficking using RNA silencing and inhibitors, we demonstrated that the early steps of endosomal trafficking are important for the intracellular transport of TYLCV in the whitefly midgut. In addition, our data show that, unlike many animal viruses, TYCLV is trafficked within cells in a manner independent of recycling endosomes, late endosomes, lysosomes, the Golgi apparatus and the endoplasmic reticulum. Instead, our results suggest that TYLCV might be transported directly from early endosomes to the basal plasma membrane and released into the hemolymph. Silencing of the sorting nexin Snx12, which may be involved in membrane tubulation, resulted in fewer viral particles in hemolymph; this suggests that the tubular endosomal network may be involved in the transport of TYLCV. Our results also support a role for the endo-lysosomal system in viral degradation. We further showed that the functions of vector early endosomes and sorting nexin Snx12 are conserved in the transmission of several other begomoviruses. Overall, our data indicate the importance of early endosomes and the tubular endosomal network in begomovirus transmission.

Transcriptome analyses suggest a novel hypothesis for whitefly adaptation to tobacco.

The adaptation of herbivorous insects to various host plants facilitates the spread and outbreak of many important invasive pests, however, the molecular mechanisms that underneath this process are poorly understood. In the past three decades, two species of the whitefly Bemisia tabaci complex, Middle East-Asia Minor 1 and Mediterranean, have invaded many countries. Their rapid and widespread invasions are partially due to their ability to infest a wide range of host plants. In this study, we determined the transcriptome and phenotypic changes of one Mediterranean whitefly population during its adaptation to tobacco, an unsuitable host plant. After several generations on tobacco, whiteflies showed increased survival and fecundity. High-throughput RNA sequencing showed that genes involved in muscle contraction and carbohydrate metabolism were significantly up-regulated after adaptation. Whiteflies reared on tobacco were further found to have increased body volume and muscle content and be trapped by tobacco trichomes in a lower frequency. On the other hand, gene expression in endosymbionts of whitefly did not change significantly after adaptation, which is consistent with the lack of cis-regulatory element on endosymbiont genomes. Over all, our data suggested that higher body volume and strengthened muscle might help whiteflies overcome physical barriers and survive on tobacco.

Sequencing and comparison of the Rickettsia genomes from the whitefly Bemisia tabaci Middle East Asia Minor I.

The whitefly, Bemisia tabaci, harbors the primary symbiont 'Candidatus Portiera aleyrodidarum' and a variety of secondary symbionts. Among these secondary symbionts, Rickettsia is the only one that can be detected both inside and outside the bacteriomes. Infection with Rickettsia has been reported to influence several aspects of the whitefly biology, such as fitness, sex ratio, virus transmission and resistance to pesticides. However, mechanisms underlying these differences remain unclear, largely due to the lack of genomic information of Rickettsia. In this study, we sequenced the genome of two Rickettsia strains isolated from the Middle East Asia Minor 1 (MEAM1) species of the B. tabaci complex in China and Israel. Both Rickettsia genomes were of high coding density and AT-rich, containing more than 1000 coding sequences, much larger than that of the coexisted primary symbiont, Portiera. Moreover, the two Rickettsia strains isolated from China and Israel shared most of the genes with 100% identity and only nine genes showed sequence differences. The phylogenetic analysis using orthologs shared in the genus, inferred the proximity of Rickettsia in MEAM1 and Rickettsia bellii. Functional analysis revealed that Rickettsia was unable to synthesize amino acids required for complementing the whitefly nutrition. Besides, a type IV secretion system and a number of virulence-related genes were detected in the Rickettsia genome. The presence of virulence-related genes might benefit the symbiotic life of the bacteria, and hint on potential effects of Rickettsia on whiteflies. The genome sequences of Rickettsia provided a basis for further understanding the function of Rickettsia in whiteflies.

Diversity and evolution of the Wolbachia endosymbionts of Bemisia (Hemiptera: Aleyrodidae) whiteflies.

Wolbachia is the most prevalent symbiont described in arthropods to date. Wolbachia can manipulate host reproduction, provide nutrition to insect hosts and protect insect hosts from pathogenic viruses. So far, 13 supergroups of Wolbachia have been identified. The whitefly Bemisia tabaci is a complex containing more than 28 morphologically indistinguishable cryptic species. Some cryptic species of this complex are invasive. In this study, we report a comprehensive survey of Wolbachia in B. tabaci and its relative B. afer from 1658 insects representing 54 populations across 13 provinces of China and one state of Australia. Based on the results of PCR or sequencing of the 16S rRNA gene, the overall rates of Wolbachia infection were 79.6% and 0.96% in the indigenous and invasive Bemisia whiteflies, respectively. We detected a new Wolbachia supergroup by sequencing five molecular marker genes including 16S rRNA, groEL, gltA, hcpA, and fbpA genes. Data showed that many protein-coding genes have limitations in detecting and classifying newly identified Wolbachia supergroups and thus raise a challenge to the known Wolbachia MLST standard analysis system. Besides, the other Wolbachia strains detected from whiteflies were clustered into supergroup B. Phylogenetic trees of whitefly mitochondrial cytochrome oxidase subunit I and Wolbachia multiple sequencing typing genes were not congruent. In addition, Wolbachia was also detected outside the special bacteriocytes in two cryptic species by fluorescence in situ hybridization, indicating the horizontal transmission of Wolbachia. Our results indicate that members of Wolbachia are far from well explored.

Transcriptomic analyses reveal the adaptive features and biological differences of guts from two invasive whitefly species.

BACKGROUND: The gut of phloem feeding insects is critical for nutrition uptake and xenobiotics degradation. However, partly due to its tiny size, genomic information for the gut of phloem feeding insects is limited. RESULTS: In this study, the gut transcriptomes of two species of invasive whiteflies in the Bemisia tabaci complex, Middle East Asia Minor 1 (MEAM1) and Mediterranean (MED), were analyzed using the Illumina sequencing. A total of 12,879 MEAM1 transcripts and 11,246 MED transcripts were annotated with a significant Blastx hit. In addition, 7,000 and 5,771 gut specific genes were respectively identified for MEAM1 and MED. Functional analyses on these gut specific genes demonstrated the important roles of gut in metabolism of insecticides and secondary plant chemicals. To reveal the molecular difference between guts of MEAM1 and MED, a comparison between gut transcriptomes of the two species was conducted and 3,910 pairs of orthologous genes were identified. Based on the ratio of nonsynonymous and synonymous substitutions, 15 genes were found evolving under positive selection. Many of those genes are predicted to be involved in metabolism and insecticide resistance. Furthermore, many genes related to detoxification were expressed at an elevated level in the gut of MED compared to MEAM1, which might be responsible for the MED's higher resistance to insecticides and environmental stresses. CONCLUSION: The sequencing of MED and MEAM1 gut transcriptomes and extensive comparisons of MEAM1 and MED gut transcripts provide substantial sequence information for revealing the role of gut in whiteflies.

The immune strategy and stress response of the Mediterranean species of the Bemisia tabaci complex to an orally delivered bacterial pathogen.

BACKGROUND: The whitefly, Bemisia tabaci, a notorious agricultural pest, has complex relationships with diverse microbes. The interactions of the whitefly with entomopathogens as well as its endosymbionts have received great attention, because of their potential importance in developing novel whitefly control technologies. To this end, a comprehensive understanding on the whitefly defense system is needed to further decipher those interactions. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a comprehensive investigation of the whitefly's defense responses to infection, via oral ingestion, of the pathogen, Pseudomonas aeruginosa, using RNA-seq technology. Compared to uninfected whiteflies, 6 and 24 hours post-infected whiteflies showed 1,348 and 1,888 differentially expressed genes, respectively. Functional analysis of the differentially expressed genes revealed that the mitogen associated protein kinase (MAPK) pathway was activated after P. aeruginosa infection. Three knottin-like antimicrobial peptide genes and several components of the humoral and cellular immune responses were also activated, indicating that key immune elements recognized in other insect species are also important for the response of B. tabaci to pathogens. Our data also suggest that intestinal stem cell mediated epithelium renewal might be an important component of the whitefly's defense against oral bacterial infection. In addition, we show stress responses to be an essential component of the defense system. CONCLUSIONS/SIGNIFICANCE: We identified for the first time the key immune-response elements utilized by B. tabaci against bacterial infection. This study provides a framework for future research into the complex interactions between whiteflies and microbes.