Plant-Derived, Nodule-Specific Cysteine-Rich Peptides as a Novel Source of Biopesticides for Controlling Citrus Greening Disease.

Nodule-specific cysteine-rich (NCR) peptides, encoded in the genome of the Mediterranean legume Medicago truncatula (barrelclover), are known to regulate plant-microbe interactions. A subset of computationally derived 20-mer peptide fragments from 182 NCR peptides was synthesized to identify those with activity against the unculturable vascular pathogen associated with citrus greening disease, 'Candidatus Liberibacter asiaticus' (CLas). Grounded in a design of experiments framework, we evaluated the peptides in a screening pipeline involving three distinct assays: a bacterial culture assay with Liberibacter crescens, a CLas-infected excised citrus leaf assay, and an assay to evaluate effects on bacterial acquisition by the nymphal stage of hemipteran vector Diaphorina citri. A subset of the 20-mer NCR peptide fragments inhibits both CLas growth in citrus leaves and CLas acquisition by D. citri. Two peptides induced higher levels of D. citri mortality. These findings reveal 20-mer NCR peptides as a new class of plant-derived biopesticide molecules to control citrus greening disease.

A Perspective on Current Therapeutic Molecule Screening Methods Against 'Candidatus Liberibacter asiaticus', the Presumed Causative Agent of Citrus Huanglongbing.

Huanglongbing (HLB), referred to as citrus greening disease, is a bacterial disease impacting citrus production worldwide and is fatal to young trees and mature trees of certain varieties. In some areas, the disease is devastating the citrus industry. A successful solution to HLB will be measured in economics: citrus growers need treatments that improve tree health, fruit production, and most importantly, economic yield. The profitability of citrus groves is the ultimate metric that truly matters when searching for solutions to HLB. Scientific approaches used in the laboratory, greenhouse, or field trials are critical to the discovery of those solutions and to estimate the likelihood of success of a treatment aimed at commercialization. Researchers and the citrus industry use a number of proxy evaluations of potential HLB solutions; understanding the strengths and limitations of each assay, as well as how best to compare different assays, is critical for decision-making to advance therapies into field trials and commercialization. This perspective aims to help the reader compare and understand the limitations of different proxy evaluation systems based on the treatment and evaluation under consideration. The researcher must determine the suitability of one or more of these metrics to identify treatments and predict the usefulness of these treatments in having an eventual impact on citrus production and HLB mitigation. As therapies advance to field trials in the next few years, a reevaluation of these metrics will be useful to guide future research efforts on strategies to mitigate HLB and vascular bacterial pathogens in other perennial crops.

An Excised Leaf Assay to Measure Acquisition of 'Candidatus Liberibacter asiaticus' by Psyllids Associated with Citrus Huanglongbing Disease.

Huanglongbing, or citrus greening disease, is the most serious disease of citrus worldwide and is associated with plant infection by 'Candidatus Liberibacter asiaticus' (CLas) and other Liberibacter species. CLas is transmitted by Diaphorina citri, the Asian citrus psyllid, in a circulative propagative manner. Circulative propagative transmission is a complex process comprising at least three steps: movement of the pathogen into vector tissues, translocation and replication of the pathogen within the vector host, and pathogen inoculation of a new host by the vector. In this work, we describe an excised leaf CLas acquisition assay, which enables precise measurements of CLas acquisition by D. citri in a streamlined laboratory assay. Briefly, healthy fourth and fifth instar D. citri nymphs acquire CLas from excised CLas-positive leaves, where the insects also complete their developmental cycle. CLas titer in the resulting adults is measured using quantitative PCR and CLas-specific 16S rRNA gene primers. We observed positive correlations between CLas titer in each leaf replicate and the CLas titer that developed in the insects after acquisition (rs = 0.78; P = 0.0002). This simple assay could be used to detect CLas acquisition phenotypes and their underlying genotypes, facilitate assessment of plant factors that impact acquisition, and screen for compounds that interfere with CLas acquisition by delivering these compounds through the excised leaf.

Host Plant Adaptation Drives Changes in Diaphorina citri Proteome Regulation, Proteoform Expression, and Transmission of 'Candidatus Liberibacter asiaticus', the Citrus Greening Pathogen.

The Asian citrus psyllid (Diaphorina citri) is a pest of citrus and the primary insect vector of the bacterial pathogen, 'Candidatus Liberibacter asiaticus' (CLas), which is associated with citrus greening disease. The citrus relative Murraya paniculata (orange jasmine) is a host plant of D. citri but is more resistant to CLas compared with all tested Citrus genotypes. The effect of host switching of D. citri between Citrus medica (citron) and M. paniculata plants on the acquisition and transmission of CLas was investigated. The psyllid CLas titer and the proportion of CLas-infected psyllids decreased in the generations after transfer from CLas-infected citron to healthy M. paniculata plants. Furthermore, after several generations of feeding on M. paniculata, pathogen acquisition (20 to 40% reduction) and transmission rates (15 to 20% reduction) in psyllids transferred to CLas-infected citron were reduced compared with psyllids continually maintained on infected citron. Top-down (difference gel electrophoresis) and bottom-up (shotgun MS/MS) proteomics methods were used to identify changes in D. citri protein expression resulting from host plant switching between Citrus macrophylla and M. paniculata. Changes in expression of insect metabolism, immunity, and cytoskeleton proteins were associated with host plant switching. Both transient and sustained feeding on M. paniculata induced distinct patterns of protein expression in D. citri compared with psyllids reared on C. macrophylla. The results point to complex interactions that affect vector competence and may lead to strategies to control the spread of citrus greening disease.

Hepatitis B virus precore/core region mutations and genotypes among hepatitis B virus chronic carriers in South-Eastern, Nigeria.

OBJECTIVES: The study aimed at detecting the prevailing hepatitis B virus (HBV) genotypes and the presence of clinically relevant mutations in the precore/core gene of the HBV DNA, among patients with chronic infection in South-eastern, Nigeria. METHODS: A total of 72 participants with chronic HBV infection were enrolled into the study. Plasma samples from those with detectable HBV DNA were subjected to nested Polymerase Chain Reaction amplification using the precore/core specific primers. This resulted to the successful amplification and sequencing of the HBV precore/core region DNA from 16 participants. Mutation analysis on the precore/core region detected the presence of certain HBV precore/core gene mutations. Genotyping was carried out by phylogenetic analysis. RESULTS: The precore region mutation at nucleotide position 1896, which is a G to A change resulting to a nonsense mutation, was detected in 6.25% of the participants. Other HBV precore region mutations that were detected include: G1899A, T1846A, G1862C, G1888A, T1821C, C1826T, A1827C, A1850T, C1858T, precore start codon Kozak sequence mutations and some novel core region mutations such as G/A1951T and G1957A. Genotyping revealed the existence of HBV genotype/subgenotype A1 (87.5%) and D (12.5%) among the participants. There was no significant difference in the occurrence of specific precore/core mutations among the HBV/hepatitis C virus dually infected and HBV mono-infected participants. CONCLUSION: The data suggest the likelihood of a more severe outcome of hepatitis caused by HBV in South-eastern Nigeria due to the occurrence of a variety of precore/core mutation, which resulted to HBeAg-negative chronic HBV infection among the participants.

Deeper Insight in Metastatic Cancer Progression; Epithelial-to- Mesenchymal Transition and Genomic Instability: Implications on Treatment Resistance.

Therapy resistance remains the major obstacle to successful cancer treatment. Epithelial-to- mesenchymal transition [EMT], a cellular reprogramming process involved in embryogenesis and organ development and regulated by a number of transcriptional factors [EMT-TFs] such as ZEB1/2, is recognized for its role in tumor progression and metastasis. Recently, a growing body of evidence has implicated EMT in cancer therapy resistance, but the actual mechanism that underlies this finding has remained elusive. For example, whether it is, the EMT states in itself or the EMT-TFs that modulate chemo or radio-resistance in cancer is still contentious. Here, we summarise the molecular mechanisms of the EMT program and chemotherapeutic resistance in cancer with specific reference to DNA damage response [DDR]. We provide an insight into the molecular interactions that exist between EMT program and DNA repair machinery in cancer and how this interaction influences therapeutic response. We review conflicting studies linking EMT and drug resistance via the DNA damage repair axis. We draw scientific evidence demonstrating how several molecular signalling, including EMT-TFs, work in operational harmony to induce EMT and confer stemness properties on the EMT-susceptible cells. We highlight the role of enhanced DNA damage repair system associated with EMT-derived stem cell-like states in promoting therapy resistance and suggest a multi-targeting modality in combating cancer treatment resistance.

Phenotypic and molecular screenings for determination of cassava mosaic disease (CMD) status in farmers' fields in Ebonyi State, Nigeria.

African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV) are among the major constraints to cassava productivity within tropical and sub-tropical regions, including Ebonyi State, Nigeria. Thus, virus indexing has become imperative to determine the status of cassava mosaic disease (CMD) in Ebonyi State, to implement appropriate preventive and control strategies. Seventy-eight cassava accessions obtained from different locations of Ebonyi State were phenotypically scored, using scales 1-5 depending on CMD symptomatic expressions, followed by multiplex-PCR and sequencing for validation. 11% of cassava accessions cultivated in Afikpo were resistant (RE) to ACMV compared to 8% of moderately-resistant (MR) accessions in Izzi and 55% of tolerant (TO) ACMV accessions in Ebonyi. 100% of cassava accessions in Onicha and 66% in Afikpo South were susceptible (SU) and highly susceptible (HS) to ACMV, respectively. With multiplex-PCR, 97.4% (ACMV) and 2.6% (EACMV) were positive. Dunn's multiple comparison tests of CMD mean incidence demonstrated differences (P < 0.05), except between RE and MR, and TO and MR. More transitions (A/G, C/T) compared to transversions (A/T, G/T), were detected, with nonsynonymous mutations (Leucine/Isoleucine; Valine/Isoleucine; Arginine/Lysine; Methionine/Isoleucine), and good bit-scores (91.13-99.07% identites; e-values of 7.00e-148-0.00e+00). Phylogeny resolved the sequences into five major groups. DNA sequencing validated the detected ACMV and EACMV species. This study revealed variants of ACMV and low adoption of RE and MR cassava accessions in the farmers' fields. The findings will guide in getting disease-free and resistant varieties as planting materials to significantly mitigate the CMD spread in Ebonyi State, Nigeria.

Genetic diversity and structure of Capsicum annuum as revealed by start codon targeted and directed amplified minisatellite DNA markers.

BACKGROUND: Identification of high resolving DNA-based markers is of paramount importance to unlock the potential of genetic diversity and selection of unique accessions of Capsicum annuum L., within Cross River and Ebonyi States of Nigeria, for breeding and conservation. Therefore, we comparatively explored the effectiveness of start codon targeted (SCoT) and directed amplified minisatellite DNA (DAMD) markers for diversity analysis of the accessions. Fifteen accessions were collected for DNA extraction and amplifications with the markers. RESULTS: Dendrograms from SCoT and DAMD categorized the accessions into five and three genetic groups, respectively, while the principal component analysis identified five genetic clusters, each from the markers. The average values of allele, gene diversity and polymorphic information content detected with SCoT and DAMD demonstrate that the two markers were effective and efficient, especially, SCoT in genetic diversity study of the accessions of pepper. Number of polymorphic loci (NPL) and percentage polymorphic loci (PPL) from SCoT (NPL = 64, PPL = 80.00-95.73%) and DAMD (NPL = 56, PPL = 53.33-86.67%) were high, but higher in SCoT markers. Other effective genetic parameters (effective number of alleles, Nei's genetic diversity and Shannon's information indices) identified with the two marker systems elucidated the allelic richness, rich genetic diversity within the populations and informative nature of the markers, especially SCoT. The intraspecific genetic diversity, interspecific genetic diversity, and coefficient of differentiation obtained with SCoT and DAMD further exposed the genetic structure with more genetic divergence within than among the populations of the accessions. Estimate of gene flow from the SCoT markers was 3.8375 and 0.6.2042 for the DAMD markers. The estimate of gene flow values from the markers indicated extensiveness with SCoT (Nm = 3.8375) and extremely extensive with DAMD (Nm = 6.2042) among the populations. CONCLUSION: This study shows that SCoT markers may be more useful and informative than DAMD in measuring genetic diversity and differentiation of the accessions of the genus Capsicum. Genetic parameters obtained with SCoT showed that the accessions from Cross River were more genetically diverse than the ones from Ebonyi State. Therefore, SCoT may be a preferred marker in evaluating genetic diversity for improvement and conservation of this spicy crop, C. capsicum.

Assessment of genetic diversity in Vigna unguiculata L. (Walp) accessions using inter-simple sequence repeat (ISSR) and start codon targeted (SCoT) polymorphic markers.

BACKGROUND: Assessment of genetic diversity of Vigna unguiculata (L.) Walp (cowpea) accessions using informative molecular markers is imperative for their genetic improvement and conservation. Use of efficacious molecular markers to obtain the required knowledge of the genetic diversity within the local and regional germplasm collections can enhance the overall effectiveness of cowpea improvement programs, hence, the comparative assessment of Inter-simple sequence repeat (ISSR) and Start codon targeted (SCoT) markers in genetic diversity of V. unguiculata accessions from different regions in Nigeria. Comparative analysis of the genetic diversity of eighteen accessions from different locations in Nigeria was investigated using ISSR and SCoT markers. DNA extraction was done using Zymogen Kit according to its manufacturer's instructions followed by amplifications with ISSR and SCoT and agarose gel electrophoresis. The reproducible bands were scored for analyses of dendrograms, principal component analysis, genetic diversity, allele frequency, polymorphic information content, and population structure. RESULTS: Both ISSR and SCoT markers resolved the accessions into five major clusters based on dendrogram and principal component analyses. Alleles of 32 and 52 were obtained with ISSR and SCoT, respectively. Numbers of alleles, gene diversity and polymorphic information content detected with ISSR were 9.4000, 0.7358 and 0.7192, while SCoT yielded 11.1667, 0.8158 and 0.8009, respectively. Polymorphic loci were 70 and 80 in ISSR and SCoT, respectively. Both markers produced high polymorphism (94.44-100%). The ranges of effective number of alleles (Ne) were 1.2887 ± 0.1797-1.7831 ± 0.2944 and 1.7416 ± 0.0776-1.9181 ± 0.2426 in ISSR and SCoT, respectively. The Nei's genetic diversity (H) ranged from 0.2112 ± 0.0600-0.4335 ± 0.1371 and 0.4111 ± 0.0226-0.4778 ± 0.1168 in ISSR and SCoT, respectively. Shannon's information index (I) from ISSR and SCoT were 0.3583 ± 0.0639-0.6237 ± 0.1759 and 0.5911 ± 0.0233-0.6706 ± 0.1604. Total gene diversity (Ht), gene diversity within population (Hs), coefficient of gene differentiation (Gst) and level of gene flow (Nm) revealed by ISSR were 0.4498, 0.3203, 0.2878 and 1.2371 respectively, while SCoT had 0.4808, 0.4522, 0.0594 and 7.9245. CONCLUSIONS: Both markers showed highest genetic diversity in accessions from Ebonyi. Our study demonstrated that SCoT markers were more efficient than ISSR for genetic diversity studies in V. unguiculata and can be integrated in the exploration of their genetic diversity for improvement and germplasm utilization.

Genomic and proteomic analysis of Schizaphis graminum reveals cyclophilin proteins are involved in the transmission of cereal yellow dwarf virus.

Yellow dwarf viruses cause the most economically important virus diseases of cereal crops worldwide and are transmitted by aphid vectors. The identification of aphid genes and proteins mediating virus transmission is critical to develop agriculturally sustainable virus management practices and to understand viral strategies for circulative movement in all insect vectors. Two cyclophilin B proteins, S28 and S29, were identified previously in populations of Schizaphisgraminum that differed in their ability to transmit the RPV strain of Cereal yellow dwarf virus (CYDV-RPV). The presence of S29 was correlated with F2 genotypes that were efficient virus transmitters. The present study revealed the two proteins were isoforms, and a single amino acid change distinguished S28 and S29. The distribution of the two alleles was determined in 12 F2 genotypes segregating for CYDV-RPV transmission capacity and in 11 genetically independent, field-collected S. graminum biotypes. Transmission efficiency for CYDV-RPV was determined in all genotypes and biotypes. The S29 isoform was present in all genotypes or biotypes that efficiently transmit CYDV-RPV and more specifically in genotypes that efficiently transport virus across the hindgut. We confirmed a direct interaction between CYDV-RPV and both S28 and S29 using purified virus and bacterially expressed, his-tagged S28 and S29 proteins. Importantly, S29 failed to interact with a closely related virus that is transported across the aphid midgut. We tested for in vivo interactions using an aphid-virus co-immunoprecipitation strategy coupled with a bottom-up LC-MS/MS analysis using a Q Exactive mass spectrometer. This analysis enabled us to identify a third cyclophilin protein, cyclophilin A, interacting directly or in complex with purified CYDV-RPV. Taken together, these data provide evidence that both cyclophilin A and B interact with CYDV-RPV, and these interactions may be important but not sufficient to mediate virus transport from the hindgut lumen into the hemocoel.