Identification of resistance to Sugarcane mosaic virus, Sugarcane streak mosaic virus, and Sugarcane bacilliform virus in new elite sugarcane accessions in India.

Sugarcane mosaic and leaf fleck diseases are significant viral diseases affecting sugarcane crops in India. The use of resistant sugarcane varieties is considered the most economical and effective approach to manage viral diseases, especially in vegetatively propagated crops such as sugarcane. Sugarcane mosaic virus (SCMV) and Sugarcane streak mosaic virus (SCSMV) are the primary pathogens responsible for mosaic disease in sugarcane-growing regions of India. Sugarcane bacilliform virus (SCBV), causing leaf fleck disease, is also often found in mixed infections with mosaic symptoms. The study aimed to identify new sources of resistance by screening sugarcane germplasm for resistance to SCMV, SCSMV, and SCBV. The screening was carried out under high inoculum using the infector row method in both plant and ratoon crops. Out of 129 genotypes tested, only 8 were found to be free of mosaic viruses, indicating a rare occurrence of resistant sources. The study revealed that mosaic disease is widespread, with nearly 95% of tested varieties/genotypes being infected with mosaic viruses. SCMV, SCSMV, and SCBV were detected in 121 out of 129 genotypes using the RT-PCR and PCR assays. Based on their response to the viruses, the tested genotypes were categorized into different resistance grades: highly resistant (grade 1), resistant (grade 2), moderately resistant (grade 3), susceptible (grade 4), and highly susceptible (grade 5). The results of the study provide valuable information about elite resistance resources that can be used for the prevention and control of mosaic disease. These resistant genotypes could also serve as potential donors for mosaic and leaf fleck disease resistance in breeding programs.

Detection of Sugarcane bacilliform virus in diseased sugarcane plants in Andhra Pradesh, India by serological and molecular methods.

Sugarcane leaf fleck incited by Sugarcane bacilliform virus is emerging as a major disease and affecting exchange of sugarcane germplasm and cultivation worldwide. Roving surveys conducted in 162 fields belonging to 81 villages spread over 14 sugarcane growing districts of Andhra Pradesh during 2021-2022 revealed 8 to 44% incidence of the disease. Mean maximum fleck disease incidence was reported in Anakapalli district (33.00%) followed by Srikakulam district (22.66%), whereas least incidence was observed in Alluri Sitharamaraju district (9.33%). The early and sensitive detection of pathogens is vital and necessary to reduce the danger of introducing new diseases or pathogen strains into sugarcane growing regions. Both serological and molecular methods were used in proposed investigation to identify the virus at the protein and nucleic acid levels. DAS-ELISA results were positive for 50 suspected SCBV infected sugarcane leaf samples out of 81, with mean absorbance (A405) values ranging from 0.50 to 2.20. Further PCR assays were performed using SCBV-specific primers targeting RT/RNase H coding region which is frequently employed as a taxonomic marker for species delineation in Badnaviruses. Out of 81 symptomatic samples collected, 61 samples gave positive results, and no amplification was observed in healthy control and negative control. Results made it evident that PCR was more sensitive than DAS-ELISA. Low virus concentration or variation in virus strains may be the reason for the low detection rate in DAS-ELISA in the current study. Extensive roving surveys conducted for the incidence of leaf fleck disease for the first time in the state of Andhra Pradesh revealed severe occurrence of leaf fleck disease under field conditions.

TDP-43 proteinopathy alters the ribosome association of multiple mRNAs including the glypican Dally-like protein (Dlp)/GPC6.

Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative disease in which 97% of patients exhibit cytoplasmic aggregates containing the RNA binding protein TDP-43. Using tagged ribosome affinity purifications in Drosophila models of TDP-43 proteinopathy, we identified TDP-43 dependent translational alterations in motor neurons impacting the spliceosome, pentose phosphate and oxidative phosphorylation pathways. A subset of the mRNAs with altered ribosome association are also enriched in TDP-43 complexes suggesting that they may be direct targets. Among these, dlp mRNA, which encodes the glypican Dally like protein (Dlp)/GPC6, a wingless (Wg/Wnt) signaling regulator is insolubilized both in flies and patient tissues with TDP-43 pathology. While Dlp/GPC6 forms puncta in the Drosophila neuropil and ALS spinal cords, it is reduced at the neuromuscular synapse in flies suggesting compartment specific effects of TDP-43 proteinopathy. These findings together with genetic interaction data show that Dlp/GPC6 is a novel, physiologically relevant target of TDP-43 proteinopathy.

Genetics of fruit yield and its component traits under different fruiting habit backgrounds in chilli (Capsicum annuum L.).

The six basic generations (two parents, F1, F2 and backcrosses) of 14 crosses developed from nine parents differing in fruits node-1 and fruit orientation were evaluated to decipher the genetics of three quantitative traits (average fruit weight, fruits plant-1 and green fruit yield plant-1) during the rainly season of 2016 and 2017. The magnitude and direction of the additive genetic effects [a], dominance genetic effects [d], magnitudes of additive genetic variance (σ2 A) and dominance genetic variance (σ2 D) varied with the genetic background of the crosses and traits. In the genetic background of crosses involving parents differing in fruit node-1, the inheritance of average fruit weight, fruits plant-1 and fruit yield plant-1 were controlled by the genes with both additive and ambidirectional dominant effects. On the contrary, genes with only additive effects controlled the inheritance of average fruit weight, fruits plant-1 and fruit yield plant-1 in most genetic backgrounds of crosses involving parents differing in fruit orientation and those differing in both fruits node-1 and fruit orientation. Further, the genes controlling the inheritance of all the traits are dispersed among the parents used in the investigation. These results are discussed in relation to strategies to be used in breeding chilli.

Structural and functional studies of Bacillus stearothermophilus serine hydroxymethyltransferase: the role of Asn(341), Tyr(60) and Phe(351) in tetrahydrofolate binding.

SHMT (serine hydoxymethyltransferase), a type I pyridoxal 5'-phosphate-dependent enzyme, catalyses the conversion of L-serine and THF (tetrahydrofolate) into glycine and 5,10-methylene THF. SHMT also catalyses several THF-independent side reactions such as cleavage of beta-hydroxy amino acids, transamination, racemization and decarboxylation. In the present study, the residues Asn(341), Tyr(60) and Phe(351), which are likely to influence THF binding, were mutated to alanine, alanine and glycine respectively, to elucidate the role of these residues in THF-dependent and -independent reactions catalysed by SHMT. The N341A and Y60A bsSHMT (Bacillus stearothermophilus SHMT) mutants were inactive for the THF-dependent activity, while the mutations had no effect on THF-independent activity. However, mutation of Phe(351) to glycine did not have any effect on either of the activities. The crystal structures of the glycine binary complexes of the mutants showed that N341A bsSHMT forms an external aldimine as in bsSHMT, whereas Y60A and F351G bsSHMTs exist as a mixture of internal/external aldimine and gem-diamine forms. Crystal structures of all of the three mutants obtained in the presence of L-allo-threonine were similar to the respective glycine binary complexes. The structure of the ternary complex of F351G bsSHMT with glycine and FTHF (5-formyl THF) showed that the monoglutamate side chain of FTHF is ordered in both the subunits of the asymmetric unit, unlike in the wild-type bsSHMT. The present studies demonstrate that the residues Asn(341) and Tyr(60) are pivotal for the binding of THF/FTHF, whereas Phe(351) is responsible for the asymmetric binding of FTHF in the two subunits of the dimer.

Importance of tyrosine residues of Bacillus stearothermophilus serine hydroxymethyltransferase in cofactor binding and L-allo-Thr cleavage.

Serine hydroxymethyltransferase (SHMT) from Bacillus stearothermophilus (bsSHMT) is a pyridoxal 5'-phosphate-dependent enzyme that catalyses the conversion of L-serine and tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. In addition, the enzyme catalyses the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids and transamination. In this article, we have examined the mechanism of the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids by SHMT. The three-dimensional structure and biochemical properties of Y51F and Y61A bsSHMTs and their complexes with substrates, especially L-allo-Thr, show that the cleavage of 3-hydroxy amino acids could proceed via Calpha proton abstraction rather than hydroxyl proton removal. Both mutations result in a complete loss of tetrahydrofolate-dependent and tetrahydrofolate-independent activities. The mutation of Y51 to F strongly affects the binding of pyridoxal 5'-phosphate, possibly as a consequence of a change in the orientation of the phenyl ring in Y51F bsSHMT. The mutant enzyme could be completely reconstituted with pyridoxal 5'-phosphate. However, there was an alteration in the lambda max value of the internal aldimine (396 nm), a decrease in the rate of reduction with NaCNBH3 and a loss of the intermediate in the interaction with methoxyamine (MA). The mutation of Y61 to A results in the loss of interaction with Calpha and Cbeta of the substrates. X-Ray structure and visible CD studies show that the mutant is capable of forming an external aldimine. However, the formation of the quinonoid intermediate is hindered. It is suggested that Y61 is involved in the abstraction of the Calpha proton from 3-hydroxy amino acids. A new mechanism for the cleavage of 3-hydroxy amino acids via Calpha proton abstraction by SHMT is proposed.

Structure determination and biochemical studies on Bacillus stearothermophilus E53Q serine hydroxymethyltransferase and its complexes provide insights on function and enzyme memory.

Serine hydroxymethyltransferase (SHMT) belongs to the alpha-family of pyridoxal 5'-phosphate-dependent enzymes and catalyzes the reversible conversion of L-Ser and tetrahydrofolate to Gly and 5,10-methylene tetrahydrofolate. 5,10-Methylene tetrahydrofolate serves as a source of one-carbon fragment in many biological processes. SHMT also catalyzes the tetrahydrofolate-independent conversion of L-allo-Thr to Gly and acetaldehyde. The crystal structure of Bacillus stearothermophilus SHMT (bsSHMT) suggested that E53 interacts with the substrate, L-Ser and tetrahydrofolate. To elucidate the role of E53, it was mutated to Q and structural and biochemical studies were carried out with the mutant enzyme. The internal aldimine structure of E53QbsSHMT was similar to that of the wild-type enzyme, except for significant changes at Q53, Y60 and Y61. The carboxyl of Gly and side chain of L-Ser were in two conformations in the respective external aldimine structures. The mutant enzyme was completely inactive for tetrahydrofolate-dependent cleavage of L-Ser, whereas there was a 1.5-fold increase in the rate of tetrahydrofolate-independent reaction with L-allo-Thr. The results obtained from these studies suggest that E53 plays an essential role in tetrahydrofolate/5-formyl tetrahydrofolate binding and in the proper positioning of Cbeta of L-Ser for direct attack by N5 of tetrahydrofolate. Most interestingly, the structure of the complex obtained by cocrystallization of E53QbsSHMT with Gly and 5-formyl tetrahydrofolate revealed the gem-diamine form of pyridoxal 5'-phosphate bound to Gly and active site Lys. However, density for 5-formyl tetrahydrofolate was not observed. Gly carboxylate was in a single conformation, whereas pyridoxal 5'-phosphate had two distinct conformations. The differences between the structures of this complex and Gly external aldimine suggest that the changes induced by initial binding of 5-formyl tetrahydrofolate are retained even though 5-formyl tetrahydrofolate is absent in the final structure. Spectral studies carried out with this mutant enzyme also suggest that 5-formyl tetrahydrofolate binds to the E53QbsSHMT-Gly complex forming a quinonoid intermediate and falls off within 4 h of dialysis, leaving behind the mutant enzyme in the gem-diamine form. This is the first report to provide direct evidence for enzyme memory based on the crystal structure of enzyme complexes.