First Report of citrus virus A in Texas associated with oak leaf patterns in Citrus sinensis

In 2018, Navarro et al. (2018a, b) identified two new negative sense coguviruses in citrus, citrus concave gum-associated virus (CCGaV) and citrus virus A (CiVA). Since then, the members of the genus Coguvirus have been also detected in other plant species (Xin et al. 2017; Wright et al. 2018; Svanella-Duma et al. 2019). In 2016, leaf flecking with oak leaf patterns were observed in five plants among embryo-rescued navel orange (NO) (Citrus sinensis (L.) Osbeck) trees grafted on C22 (C. sunki x Poncirus trifoliata) rootstock, maintained in a shade house. Madam Vinous (MV) sweet orange trees graft-inoculated with blind buds from the symptomatic NO plants developed the same symptoms in the new growth. These symptoms were similar to those on the citrus concave gum (CG) source tree of the California isolate CG301, one of the standard citrus disease isolates used as a positive control for biological indexing (Roistacher et al. 2000). None of the trees with oak leaf symptoms tested positive with reverse transcription (RT)-PCR for a panel of viruses and viroids commonly infecting citrus (Table S1) . In this study, CG301 leaf RNA-Seq data was used as a platform to identify any viral agent(s) associated with the oak leaf symptoms observed in the symptomatic NO trees (Fig. S1). Of ~162.8 million paired-end CG301 RNA-Seq reads (150 bp), de novo assembly of ~9.6 million reads, not mapped to C. sinensis genome (v.1.1), yielded 5,375 contigs. BLASTn using NCBI virus database (txid 10239) identified two contigs, #49 (6,715 nt) and #20 (2,764 nt), which exhibited ~96% sequence identity, respectively, to RNA1 and 2 of CiVA isolate W4 (MG764565; MG764566) and 71-73% identity to that of CCGaV isolate CGW2 (KX960112; KX960111). 5'-Nuclease assay developed based on contig #20 detected coguviral sequences in the five symptomatic NO and graft-inoculated MV trees as well as in CG301 but not in 44 asymptomatic field trees located near the shade house where the symptomatic NO trees had been kept. A full genomic sequence of the coguviruses present in CG301 and a symptomatic NO tree was reconstructed by RT-PCR. Both CG301 and NO isolate have a 6689 nt long negative sense RNA1 (MT922052; MK689372) encoding RNA-dependent RNA polymerase (RdRp) and a 2739 nt long ambisense RNA2 (MT922053; MK689373) encoding movement protein (MP) and nucleocapsid protein (NP). The isolate CG301 and NO share ~96% nucleotide sequence identity. The genome of both CG301 and NO isolate share 95.4-97.8 % sequence identity to that of CiVA isolate W4 and 70-72.9 % sequence identity to CCGaV isolate CGW2. BLASTp showed that RdRp of CG301 and NO isolate have 96.3-97.7 % sequence identity to CiVA W4 RdRp and ~77 % to CCGaV CGW2 RdRp. These data indicated the presence of CiVA in the symptomatic NO trees and in the concave gum source tree CG301. Recent reports of CiVA in South Africa and Greece indicated a potential wider distribution of CiVA in various citrus growing regions that may be associated with two graft-transmissible citrus diseases, citrus concave gum and impietratura disease (Roistacher et al. 2000; Velázquez et al. 2019; Beris et al. 2021; Bester et al. 2021). Although the source of CiVA in the symptomatic NO trees and the degree of CiVA prevalence in Texas had not been determined yet, a possible involvement of vector(s) or other means of spread (e.g. seed transmission) cannot be ruled out (Timmer et al. 2017). The current study demonstrated the need for further studies to determine the level of threat of coguviruses for citrus production in Texas.

Complete genomic sequence and phylogenomics analysis of Agrobacterium strain AB2/73: a new Rhizobium species with a unique mega-Ti plasmid

Background The Agrobacterium strain AB2/73 has a unique host range for the induction of crown gall tumors, and contains an exceptionally large, over 500 kbp mega Ti plasmid. We used whole genome sequencing to fully characterize and comparatively analyze the complex genome of strain AB2/73, including its Ti plasmid and virulence factors. Results We obtained a high-quality, full genomic sequence of AB2/73 by a combination of short-read Illumina sequencing and long-read Nanopore sequencing. The AB2/73 genome has a total size of 7,266,754 bp with 59.5% GC for which 7012 genes (6948 protein coding sequences) are predicted. Phylogenetic and comparative genomics analysis revealed that strain AB2/73 does not belong to the genus Agrobacterium, but to a new species in the genus Rhizobium, which is most related to Rhizobium tropici. In addition to the chromosome, the genome consists of 6 plasmids of which the largest two, of more than 1 Mbp, have chromid-like properties. The mega Ti plasmid is 605 kbp in size and contains two, one of which is incomplete, repABC replication units and thus appears to be a cointegrate consisting of about 175 kbp derived from an unknown Ti plasmid linked to 430 kbp from another large plasmid. In pTiAB2/73 we identified a complete set of virulence genes and two T-DNAs. Besides the previously described T-DNA we found a larger, second T-DNA containing a 6b-like onc gene and the acs gene for agrocinopine synthase. Also we identified two clusters of genes responsible for opine catabolism, including an acc-operon for agrocinopine degradation, and genes putatively involved in ridéopine catabolism. The plasmid also harbours tzs, iaaM and iaaH genes for the biosynthesis of the plant growth regulators cytokinin and auxin. Conclusions The comparative genomics analysis of the high quality genome of strain AB2/73 provided insight into the unusual phylogeny and genetic composition of the limited host range Agrobacterium strain AB2/73. The description of its unique genomic composition and of all the virulence determinants in pTiAB2/73 will be an invaluable tool for further studies into the special host range properties of this bacterium.

Complete Genomic Sequence and Phylogenomics Analysis of Agrobacterium Strain AB2/73: A New Rhizobium Species with a Unique mega-Ti Plasmid

Background: The Agrobacterium strain AB2/73 has a unique host range for the induction of crown gall tumors, and contains an exceptionally large, over 500 kbp mega Ti plasmid. We used whole genome sequencing to fully characterize and comparatively analyze the complex genome of strain AB2/73, including its Ti plasmid and virulence factors. Results: We obtained a high-quality, full genomic sequence of AB2/73 by a combination of short-read Illumina sequencing and long-read Nanopore sequencing. The AB2/73 genome has a total size of 7,266,754 bp with 59.5% GC for which 7,012 genes (6,948 protein coding sequences) are predicted. Phylogenetic and comparative genomics analysis revealed that strain AB2/73 does not belong to the genus Agrobacterium, but to a new species in the genus Rhizobium, which is most related to Rhizobium tropici. In addition to the chromosome, the genome consists of 6 plasmids of which the largest two, of more than 1 Mbp, have chromid-like properties. The mega Ti plasmid is 605 kbp in size and contains two, one of which is incomplete, repABC replication units and thus appears to be a cointegrate consisting of about 175 kbp derived from an unknown Ti plasmid linked to 430 kbp from another large plasmid. In pTiAB2/73 we identified a complete set of virulence genes and two T-DNAs. Besides the previously described T-DNA we found a larger, second T-DNA containing a 6b-like onc gene and the acs gene for agrocinopine synthase. Also we identified two clusters of genes responsible for opine catabolism, including an acc-operon for agrocinopine degradation, and genes putatively involved in ridéopine catabolism. The plasmid also harbours tzs, iaaM and iaaH genes for the biosynthesis of the plant growth regulators cytokinin and auxin. Conclusions: The comparative genomics analysis of the high quality genome of strain AB2/73 provided insight into the unusual phylogeny and genetic composition of the limited host range Agrobacterium strain AB2/73. The description of its unique genomic composition and of all the virulence determinants in pTiAB2/73 will be an invaluable tool for further studies into the special host range properties of this bacterium.

Exploring the Effect of Climate Factors on SNPs within FHA Domain Genes in Eurasian Arabidopsis Ecotypes

The rapid developments in high-throughput sequencing technologies have allowed researchers to analyze the full genomic sequence of organisms faster and cheaper than ever before. An important application of such advancements is to identify the impact of single nucleotide polymorphisms (SNPs) on the phenotypes and genotypes of the same species by discovering the factors that affect the occurrence of SNPs. The focus of this study is to determine whether climate factors such as the main climate, the precipitation, and the temperature affecting a certain geographical area might be associated with specific variations in certain ecotypes of the plant Arabidopsis thaliana. To test our hypothesis we analyzed 18 genes that encode Forkhead-Associated domain-containing proteins. They were extracted from 80 genomic sequences gathered from within 8 Eurasian regions. We used k-means clustering to separate the plants into distinct groups and evaluated the clusters using an innovative scoring system based upon the Köppen-Geiger climate classification system. The methods we used allow the selection of candidate clusters most likely to contain samples with similar polymorphisms. These clusters show that there is a correlation between genomic variations and the geographic distribution of those ecotypes.

A Comprehensive Analysis of cis-Acting RNA Elements in the SARS-CoV-2 Genome by a Bioinformatics Approach

The emergence of a new coronavirus (CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for severe respiratory disease in humans termed coronavirus disease of 2019 (COVID-19), became a new global threat for health and the economy. The SARS-CoV-2 genome is about a 29,800-nucleotide-long plus-strand RNA that can form functionally important secondary and higher-order structures called cis-acting RNA elements. These elements can interact with viral proteins, host proteins, or other RNAs and be involved in regulating translation and replication processes of the viral genome and encapsidation of the virus. However, the cis-acting RNA elements and their biological roles in SARS-CoV-2 as well as their comparative analysis in the closely related viral genome have not been well explored, which is very important to understand the molecular mechanism of viral infection and pathogenies. In this study, we used a bioinformatics approach to identify the cis-acting RNA elements in the SARS-CoV-2 genome. Initially, we aligned the full genomic sequence of six different CoVs, and a phylogenetic analysis was performed to understand their evolutionary relationship. Next, we predicted the cis-acting RNA elements in the SARS-CoV-2 genome using the structRNAfinder tool. Then, we annotated the location of these cis-acting RNA elements in different genomic regions of SARS-CoV-2. After that, we analyzed the sequence conservation patterns of each cis-acting RNA element among the six CoVs. Finally, the presence of cis-acting RNA elements across different CoV genomes and their comparative analysis was performed. Our study identified 12 important cis-acting RNA elements in the SARS-CoV-2 genome; among them, Corona_FSE, Corona_pk3, and s2m are highly conserved across most of the studied CoVs, and Thr_leader, MAT2A_D, and MS2 are uniquely present in SARS-CoV-2. These RNA structure elements can be involved in viral translation, replication, and encapsidation and, therefore, can be potential targets for better treatment of COVID-19. It is imperative to further characterize these cis-acting RNA elements experimentally for a better mechanistic understanding of SARS-CoV-2 infection and therapeutic intervention.

Emergence of a Multidrug-Resistant Enterobacter hormaechei Clinical Isolate from Egypt Co-Harboring mcr-9 and blaVIM-4

This study describes the first full genomic sequence of an mcr-9 and blaVIM-4-carrying multidrug-resistant Enterobacter hormaechei clinical isolate from Egypt. The strain was isolated in April 2015 from the sputum of a patient in Cairo, Egypt. The mcr-9 and blaVIM-4 genes were identified by PCR screening and DNA sequencing; the isolate was subjected to antimicrobial susceptibility testing, conjugation experiments, and whole genomic sequencing. mcr-9 and blaVIM-4 were carried by an IncHI2 plasmid, pAMS-38a (281,121 bp in size); the plasmid also carried genes conferring resistance against sulfonamides (sul1), quinolones (qnrA1), trimethoprim (dfrA1), β-lactams (blaTEM-1B), aminoglycosides (aac (6’)-II, aadA23, aadA2b, and ant(2’’)-Ia). The strain was susceptible to colistin (MIC, <0.25 μg/mL); this could be due to the absence of the qseC/qseB regulatory system located downstream of mcr-9 in Enterobacterales, which is involved in the induction of colistin-resistance. The genetic context of mcr-9 and blaVIM-4 was identified as IS1-mcr-9-IS903-pcoS-∆pcoE-rcnA and intI1-blaVIM-4—aac (6’)-II-dfrA1-∆aadA23-smr-ISPa21-qacE∆1, respectively. This is the first report of an mcr-9 and blaVIM-4 /IncHI2-carrying multidrug-resistant E. hormaechei clinical isolate from Africa and the Middle East. Plasmids of the IncHI2 group and the two insertion sequences (IS1, and IS903) might be the main vehicles for dissemination of mcr-9. Further screening for mcr-9 is essential for identifying its incidence and to prevent its dissemination.

Visualizing Sacbrood Virus of Honey Bees via Transformation and Coupling with Enhanced Green Fluorescent Protein

Sacbrood virus (SBV) of honey bees is a picornavirus in the genus Iflavirus. Given its relatively small and simple genome structure, single positive-strand RNA with only one ORF, cloning the full genomic sequence is not difficult. However, adding nonsynonymous mutations to the bee iflavirus clone is difficult because of the lack of information about the viral protein processes. Furthermore, the addition of a reporter gene to the clones has never been accomplished. In preliminary trials, we found that the site between 3′ untranslated region (UTR) and poly(A) can retain added sequences. We added enhanced green fluorescent protein (EGFP) expression at this site, creating a SBV clone with an expression tag that does not affect virus genes. An intergenic region internal ribosome entry site (IRES) from Black queen cell virus (BQCV) was inserted to initiate EGFP expression. The SBV-IRES-EGFP clone successfully infected Apis cerana and Apis mellifera, and in A. cerana larvae, it was isolated and passaged using oral inoculation. The inoculated larvae had higher mortality and the dead larvae showed sacbrood symptoms. The added IRES-EGFP remained in the clone through multiple passages and expressed the expected EGFP in all infected bees. We demonstrated the ability to add gene sequences in the site between 3′-UTR and poly(A) in SBV and the potential to do so in other bee iflaviruses; however, further investigations of the mechanisms are needed. A clone with a desired protein expression reporter will be a valuable tool in bee virus studies.

Evolution of the Autism-Associated Neuroligin-4 Gene Reveals Broad Erosion of Pseudoautosomal Regions in Rodents

Variants in genes encoding synaptic adhesion proteins of the neuroligin family, most notably neuroligin-4, are a significant cause of autism spectrum disorders in humans. Although human neuroligin-4 is encoded by two genes, NLGN4X and NLGN4Y, that are localized on the X-specific and male-specific regions of the two sex chromosomes, the chromosomal localization and full genomic sequence of the mouse Nlgn4 gene remain elusive. Here, we analyzed the neuroligin-4 genes of numerous rodent species by direct sequencing and bioinformatics, generated complete drafts of multiple rodent neuroligin-4 genes, and examined their evolution. Surprisingly, we find that the murine Nlgn4 gene is localized to the pseudoautosomal region (PAR) of the sex chromosomes, different from its human orthologs. We show that the sequence differences between various neuroligin-4 proteins are restricted to hotspots in which rodent neuroligin-4 proteins contain short repetitive sequence insertions compared with neuroligin-4 proteins from other species, whereas all other protein sequences are highly conserved. Evolutionarily, these sequence insertions initiate in the clade eumuroidea of the infraorder myomorpha and are additionally associated with dramatic changes in noncoding sequences and gene size. Importantly, these changes are not exclusively restricted to neuroligin-4 genes but reflect major evolutionary changes that substantially altered or even deleted genes from the PARs of both sex chromosomes. Our results show that despite the fact that the PAR in rodents and the neuroligin-4 genes within the rodent PAR underwent massive evolutionary changes, neuroligin-4 proteins maintained a highly conserved core structure, consistent with a substantial evolutionary pressure preserving its physiological function.