Molecular Characterization of \u3ci\u3eCitrus tatter leaf virus\u3c/i\u3e Historically Associated with Meyer Lemon Trees: Complete Genome Sequence and Development of Biologically Active In Vitro Transcripts

Citrus tatter leaf virus isolated from Meyer lemon trees (CTLV-ML) from California and Florida induces bud union incompatibility of citrus trees grafted on the widely used trifoliate and trifoliate hybrid rootstocks. The complete genome sequence of CTLV-ML was determined to be 6,495 nucleotides (nts), with two overlapping open reading frames (ORFs) and a poly (A) tail at the 3′ end. The genome organization is similar to other capilloviruses, with ORF1 (nts 37 to 6,354) encoding a putative 242-kDa polyprotein which contains replication-associated domains plus a coat protein (CP), and ORF2 (nts 4,788 to 5,750), which is located within ORF1 in a different reading frame and encodes a putative movement protein. Although the proteins encoded by CTLV-ML possesses 84 to 96% amino acid sequence identity with strains of Apple stem grooving virus (ASGV), we observed two strikingly different regions in ORF1: variable region I (amino acids 532 to 570) and variable region II (amino acids 1,583 to 1,868), with only 15 to 18 and 56 to 62% identities, respectively, with the corresponding regions of ASGV strains. Conditions for a herbaceous systemic assay host were optimized in which the wildtype virus induced systemic infection in Phaseolus vulgaris cv. Light Red Kidney (LRK) bean plants at 19 or 22°C but not at higher temperatures. In vitro transcripts generated from full-length cDNA clones induced systemic symptoms on LRK bean plants similar to that of the wild-type virus. Replication of the recombinant virus was confirmed by hybridization of a 5′ positive-stranded RNA-specific probe to a genome-sized RNA and by reverse-transcription polymerase chain reaction

Characterization of the 5′- and 3′-Terminal Subgenomic RNAs Produced by a Capillovirus: Evidence for a CP Subgenomic RNA

The members of Capillovirus genus encode two overlapping open reading frames (ORFs): ORF1 encodes a large polyprotein containing the replication-associated proteins plus a coat protein (CP), and ORF2 encodes a movement protein (MP), located within ORF1 in a different reading frame. Organization of the CP sequence as part of the replicase ORF is unusual in capilloviruses. In this study, we examined the capillovirus genome expression strategy by characterizing viral RNAs produced by Citrus tatter leaf virus (CTLV), isolate ML, a Capillovirus. CTLV-ML produced a genome-length RNA of ∼6.5-kb and two 3′-terminal sgRNAs in infected tissue that contain the MP and CP coding sequences (3′-sgRNA1), and the CP coding sequence (3′-sgRNA2), respectively. Both 3′-sgRNAs initiate at a conserved octanucleotide (UUGAAAGA), and are 1826 (3′-sgRNA1) and 869 (3′-sgRNA2) nts with 119 and 15 nt leader sequences, respectively, suggesting that these two 3′- sgRNAs could serve to express the MP and CP. Additionally, accumulation of two 5′-terminal sgRNAs of 5586 (5′-sgRNA1) and 4625 (5′-sgRNA2) nts was observed, and their 3′-termini mapped to 38–44 nts upstream of the transcription start sites of 3′-sgRNAs. The presence of a separate 3′-sgRNA corresponding to the CP coding sequence and its cognate 5′-terminal sgRNA (5′-sgRNA1) suggests that CTLV-ML produces a dedicated sg mRNA for the expression of its CP

The pathogenicity determinant of \u3ci\u3eCitrus tristeza virus\u3c/i\u3e causing the seedling yellows syndrome maps at the 3′-terminal region of the viral genome

Citrus tristeza virus (CTV) (genus Closterovirus, family Closteroviridae) causes some of the more important viral diseases of citrus worldwide. The ability to map disease-inducing determinants of CTV is needed to develop better diagnostic and disease control procedures. A distinctive phenotype of some isolates of CTV is the ability to induce seedling yellows (SY) in sour orange, lemon and grapefruit seedlings. In Florida, the decline isolate of CTV, T36, induces SY, whereas a widely distributed mild isolate, T30, does not. To delimit the viral sequences associated with the SY syndrome, we created a number of T36/T30 hybrids by substituting T30 sequences into different regions of the 3′ half of the genome of an infectious cDNA of T36. Eleven T36/T30 hybrids replicated in Nicotiana benthamiana protoplasts. Five of these hybrids formed viable virions that were mechanically transmitted to Citrus macrophylla, a permissive host for CTV. All induced systemic infections, similar to that of the parental T36 clone. Tissues from these C. macrophylla source plants were then used to graft inoculate sour orange and grapefruit seedlings. Inoculation with three of the T30/T36 hybrid constructs induced SY symptoms identical to those of T36; however, two hybrids with T30 substitutions in the p23-3′ nontranslated region (NTR) (nucleotides 18 394–19 296) failed to induce SY. Sour orange seedlings infected with a recombinant non-SY p23-3′ NTR hybrid also remained symptomless when challenged with the parental virus (T36), demonstrating the potential feasibility of using engineered constructs of CTV to mitigate disease.mp

Contribution of Microbe-Mediated Processes in Nitrogen Cycle to Attain Environmental Equilibrium

Nitrogen (N), the most important element, is required by all living organisms for the synthesis of complex organic molecules like amino acids, proteins, lipids etc. Nitrogen cycle is considered to be the most complex yet arguably important cycle next to carbon cycle. Nitrogen cycle includes oxic and anoxic reactions like organic N mineralization, ammonia assimilation, nitrification denitrification, anaerobic ammonium oxidation (anammox), dissimilatory nitrate reduction to ammonium (DNRA), comammox, codenitrification etc. Nitrogen cycling is one of the most crucial processes required for the recycling of essential chemical requirements on the planet. Soil microorganisms not only improve N-cycle balance but also pave the way for sustainable agricultural practices, leading to improved soil properties and crop productivity as most plants are opportunistic in the uptake of soluble or available forms of N from soil. Microbial N transformations are influenced by plants to improve their nutrition and vice versa. Diverse microorganisms, versatile metabolic activities, and varied biotic and abiotic conditions may result in the shift in the equilibrium state of different N-cycling processes. This chapter is an overview of the mechanisms and genes involved in the diverse microorganisms associated in the operation of nitrogen cycle and the roles of such microorganisms in different agroecosystems

Heterologous minor coat proteins of \u3ci\u3eCitrus tristeza virus\u3c/i\u3e strains affect encapsidation, but the coexpression of HSP70h and p61 restores encapsidation to wild-type levels

The long flexuous bipolar virions of Citrus tristeza virus (CTV), a Closterovirus, are encapsidated with two capsid proteins at opposite ends: the minor coat protein (CPm) encapsidates the 5′ 630 nts of the genomic RNA and the major coat protein encapsidates the remainder of the genome. In this study, we found encapsidation of CTV CPm in the absence of other assembly-related proteins is highly specific in contrast to most plant viruses that allow virion assembly by a range of heterologous coat proteins. Heterologous CPms with 95–96% amino acid identity from related strains in CTV-CPm, a replicon with CPm as the only assemblyrelated ORF, either failed to initiate encapsidation or reduced encapsidation substantially. Substitution of subsets of amino acids revealed that the amino acids that differ between positions 121 and 180 of the VT strain, and 61 and 120 of the T3 strain were involved in specific encapsidation. We further mapped the specific encapsidation to a single amino acid: mutation of methionine165 to threonine (VT type) or serine105 to proline (T3 type) in CTV-CPm failed to form nucleocapsids. However, the heterologous CPm in combination with both HSP70h and p61 proteins, but not HSP70h or p61 alone, encapsidated at wild-type levels, suggesting that specific encapsidation by CPm was mitigated by the combination of HSP70h and p61. Thus, in addition to the previously described functions of HSP70h and p61 of greatly enhanced virion formation and restriction of CPm encapsidation to the 5′ 630 nts of the genomic RNA, these proteins facilitate encapsidation by heterologous CPms

Closterovirus bipolar virion: Evidence for initiation of assembly by minor coat protein and its restriction to the genomic RNA 5\u27 region

The long flexuous virions of the Closteroviridae have a unique bipolar architecture incorporating two coat proteins, with most of the helical nucleocapsid encapsidated by the major coat protein (CP) and a small portion of one end encapsidated by the minor coat protein (CPm). It is not known whether CPm encapsidates the genomic RNA and, if so, which end and what effects transition between the two coat proteins. Two other virus-encoded proteins, an HSP70 homolog (HSP70h) and an = 61-kDa protein, are required to augment virion assembly. In this work, we examine the in vivo encapsidation of Citrus tristeza virus by its CPm in the absence of CP. In the absence of other assembly-related proteins, CPm protected a family of 5\u27 coterminal RNAs, apparently because of pausing at different locations along the genomic RNA. Most of the nucleocapsids formed by CPm were short, but a few were fulllength and infectious. Mutations within the 5\u27 nontranslated region demonstrated that the CPm origin of assembly overlaps the previously described conserved stem-and-loop structures that function as a cis-acting element required for RNA synthesis. Thus, in the absence of CP, the CPm encapsidation is initiated from the 5\u27 end of the genomic RNA. Coexpression of HSP70h and the p61 protein with CPm in protoplasts restricted encapsidation to the 5\u27 = 630 nucleotides, which is close to the normal boundary of the bipolar virion, whereas the presence of either HSP70h or the p61 protein alone did not limit encapsidation by CPm

A Simple Method of Obtaining the Band Strengths in the Electronic Spectra of Diatomic Molecules

Relative band strengths of diatomic molecules for which the product of Franck-Condon factor and r-centroid is approximately equal to 1 for (0,0) band can be determined by a simple method which will be in good agreement with the smoothed array of experimental values