Micronutrients Affect Expression of Induced Resistance Genes in Hydroponically Grown Watermelon against Fusarium oxysporum f. sp. niveum and Meloidogyne incognita

The soil-borne pathogens, particularly Fusarium oxysporum f. sp. niveum (FON) and southern root-knot nematode (RKN, Meloidogyne incognita) are the major threats to watermelon production in the southeastern United States. The role of soil micronutrients on induced resistance (IR) to plant diseases is well-documented in soil-based media. However, soil-based media do not allow us to determine the contribution of individual micronutrients in the induction of IR. In this manuscript, we utilized hydroponics-medium to assess the effect of controlled application of micronutrients, including iron (Fe), manganese (Mn), and zinc (Zn) on the expression of important IR genes (PR1, PR5, and NPR1 from salicylic acid (SA) pathway, and VSP, PDF, and LOX genes from jasmonic acid (JA) pathway) in watermelon seedlings upon inoculation with either FON or RKN or both. A subset of micronutrient-treated plants was inoculated (on the eighth day of micronutrient application) with FON and RKN (single or mixed inoculation). The expression of the IR genes in treated and control samples was evaluated using qRT-PCR. Although, significant phenotypic differences were not observed with respect to the severity of wilt symptoms or RKN galling with any of the micronutrient treatments within the 30-day experimental period, differences in the induction of IR genes were considerably noticeable. However, the level of gene expression varied with sampling period, type and concentration of micronutrients applied, and pathogen inoculation. In the absence of pathogens, micronutrient applications on the seventh day, in general, downregulated the expression of the majority of the IR genes. However, pathogen inoculation preferentially either up- or down-regulated the expression levels of the IR genes at three days post-inoculation depending on the type and concentration of micronutrients. The results demonstrated here indicate that micronutrients in watermelon may potentially make watermelon plants susceptible to infection by FON and RKN. However, upon infection the IR genes are significantly up-regulated that they may potentially aid the prevention of further infection via SA- and JA-pathways. This is the first demonstration of the impact of micronutrients affecting IR in watermelon against FON and RKN infection.

Polymorphic landscape of SARS-CoV-2 genomes isolated from Indian population in 2020 demonstrates rapid evolution in ORF3a, ORF8, nucleocapsid phosphoprotein and spike glycoprotein.

India, with around 15 million COVID-19 cases, recently became the second worst-hit nation by the SARS-CoV-2 pandemic. In this study, we analyzed the mutation and selection landscape of 516 unique and complete genomes of SARS-CoV-2 isolates from India in a 12-month span (from Jan to Dec 2020) to understand how the virus is evolving in this geographical region. We identified 953 genome-wide loci displaying single nucleotide polymorphism (SNP) and the Principal Component Analysis and mutation plots of the datasets indicate an increase in genetic variance with time. The 42% of the polymorphic sites display substitutions in the third nucleotide position of codons indicating that non-synonymous substitutions are more prevalent. These isolates displayed strong evidence of purifying selection in ORF1ab, spike, nucleocapsid, and membrane glycoprotein. We also find some evidence of localized positive selections ORF1ab, spike glycoprotein, and nucleocapsid. The CDSs for ORF3a, ORF8, nucleocapsid phosphoprotein, and spike glycoprotein were found to evolve at rapid rate. This study will be helpful in understanding the dynamics of rapidly evolving SARS-CoV-2.

Biochemistry of plants N-heterocyclic non-protein amino acids.

Plants catalyze the biosynthesis of a large number of non-protein amino acids, which are usually toxic for other organisms. In this review, the chemistry and metabolism of N-heterocyclic non-protein amino acids from plants are described. These N-heterocyclic non-protein amino acids are composed of ß-substituted alanines and include mimosine, ß-pyrazol-1-yl-L-alanine, willardiine, isowillardiine, and lathyrine. These ß-substituted alanines consisted of an N-heterocyclic moiety and an alanyl side chain. This review explains how these individual moieties are derived from their precursors and how they are used as the substrate for biosynthesizing the respective N-heterocyclic non-protein amino acids. In addition, known catabolism and possible role of these non-protein amino acids in the actual host is explained.

Insights into high mobility group A (HMGA) proteins from Poaceae family: An in silico approach for studying homologs.

High mobility group (HMG) proteins are the major architectural proteins. Among HMG proteins, High Mobility Group A (HMGA) is characterized by AT-hook (ATH) motifs, which have an affinity for AT-rich DNA. In this study, we characterized the plant HMGAs from the Poaceae family using in silico methods. The protein sequences for rice HMGAs were retrieved and the corresponding orthologs from grasses were extracted. The phylogenetic analysis identified three major evolutionary clades of grass HMGAs and their ATH motif analysis revealed that HMGAs from clade 1 and 2, except for clade 2 HMGAs, are devoid of high-affinity DNA-binding domain. The clade 2 HMGAs also displayed a highly conserved length of all the spacers and the length of the C-terminal tail following the last ATH. Moreover, the C-terminal tail in clade 2 HMGAs is smaller than HMGAs from any other clade. Unlike clade 2, other clades of Poaceae HMGAs displayed high variability in the length of spacers. Despite several differences among HMGAs of different clades in Poaceae, the H1/H5 domain was found to be highly conserved. This study has revealed the detailed analyses of Poaceae HMGAs and it will be useful for further investigation aiming at the determination of precise biological functions and molecular mechanisms of grass HMGAs.

Heterologous Expression and Characterization of Mimosinase from Leucaena leucocephala.

Heterologous expression of eukaryotic genes in bacterial system is an important method in synthetic biology to characterize proteins. It is a widely used method, which can be sometimes quite challenging, as a number of factors that act along the path of expression of a transgene to mRNA, and mRNA to protein, can potentially affect the expression of a transgene in a heterologous system. Here, we describe a method for successful cloning and expression of mimosinase-encoding gene from Leucaena leucocephala (leucaena) in E. coli as the heterologous host. Mimosinase is an important enzyme especially in the context of metabolic engineering of plant secondary metabolite as it catalyzes the degradation of mimosine, which is a toxic secondary metabolite found in all Leucaena and Mimosa species. We also describe the methods used for characterization of the recombinant mimosinase.

An O-acetylserine (thiol) lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthase.

In plants, the final step of cysteine formation is catalyzed by O-acetylserine (thiol) lyase (OAS-TL). The purpose of this study was to isolate and characterize an OAS-TL from the tree legume Leucaena leucocephala (leucaena). Leucaena contains a toxic, nonprotein amino acid, mimosine, which is also formed by an OAS-TL, and characterization of this enzyme is essential for developing a mimosine-free leucaena for its use as a protein-rich fodder. The cDNA for a cytosolic leucaena OAS-TL isoform was obtained through interspecies suppression subtractive hybridization. A 40-kDa recombinant protein was purified from Escherichia coli and used in enzyme activity assays where it was found to synthesize only cysteine. The enzyme followed Michaelis-Menten kinetics, and the Km was calculated to be 1,850±414 µM sulfide and the Vmax was 200.6±19.92 µM cysteine min(-1). The N-terminal affinity His-tag was cleaved from the recombinant OAS-TL to eliminate its possible interference in binding with the substrate, 3-hydroxy-4-pyridone, for mimosine formation. The His-tag-cleaved OAS-TL was again observed to catalyze the formation of cysteine but not mimosine. Thus, the cytosolic OAS-TL from leucaena used in this study is specific for only cysteine synthesis and is different from previously reported OAS-TLs that also function as ß-substituted alanine synthases.

A carbon-nitrogen lyase from Leucaena leucocephala catalyzes the first step of mimosine degradation.

The tree legume Leucaena leucocephala contains a large amount of a toxic nonprotein aromatic amino acid, mimosine, and also an enzyme, mimosinase, for mimosine degradation. In this study, we isolated a 1,520-bp complementary DNA (cDNA) for mimosinase from L. leucocephala and characterized the encoded enzyme for mimosine-degrading activity. The deduced amino acid sequence of the coding region of the cDNA was predicted to have a chloroplast transit peptide. The nucleotide sequence, excluding the sequence for the chloroplast transit peptide, was codon optimized and expressed in Escherichia coli. The purified recombinant enzyme was used in mimosine degradation assays, and the chromatogram of the major product was found to be identical to that of 3-hydroxy-4-pyridone (3H4P), which was further verified by electrospray ionization-tandem mass spectrometry. The enzyme activity requires pyridoxal 5'-phosphate but not α-keto acid; therefore, the enzyme is not an aminotransferase. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products. The dependence of the enzyme on pyridoxal 5'-phosphate and the production of 3H4P with the release of ammonia indicate that it is a carbon-nitrogen lyase. It was found to be highly efficient and specific in catalyzing mimosine degradation, with apparent Km and Vmax values of 1.16×10(-4) m and 5.05×10(-5) mol s(-1) mg(-1), respectively. The presence of other aromatic amino acids, including l-tyrosine, l-phenylalanine, and l-tryptophan, in the reaction did not show any competitive inhibition. The isolation of the mimosinase cDNA and the biochemical characterization of the recombinant enzyme will be useful in developing transgenic L. leucocephala with reduced mimosine content in the future.

midD-encoded 'rhizomimosinase' from Rhizobium sp. strain TAL1145 is a C-N lyase that catabolizes L-mimosine into 3-hydroxy-4-pyridone, pyruvate and ammonia.

Rhizobium sp. strain TAL1145 catabolizes mimosine, which is a toxic non-protein amino acid present in Leucaena leucocephala (leucaena). The objective of this investigation was to study the biochemical and catalytic properties of the enzyme encoded by midD, one of the TAL1145 genes involved in mimosine degradation. The midD-encoded enzyme, MidD, was expressed in Escherichia coli, purified and used for biochemical and catalytic studies using mimosine as the substrate. The reaction products in the enzyme assay were analyzed by HPLC and mass spectrometry. MidD has a molecular mass of ~45 kDa and its catalytic activity was found to be optimal at 37 °C and pH 8.5. The major product formed in the reaction had the same retention time as that of synthetic 3-hydroxy-4-pyridone (3H4P). It was confirmed to be 3H4P by MS/MS analysis of the HPLC-purified product. The K m, V max and K cat of MidD were 1.27 × 10(-4) mol, 4.96 × 10(-5) mol s(-1) mg(-1), and 2,256.05 s(-1), respectively. Although MidD has sequence similarities with aminotransferases, it is not an aminotransferase because it does not require a keto acid as the co-substrate in the degradation reaction. It is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and the addition of 50 µM hydroxylamine completely inhibited the reaction. However, the supplementation of the reaction with 0.1 µM PLP restored the catalytic activity of MidD in the reaction containing 50 µM hydroxylamine. The catalytic activity of MidD was found to be specific to mimosine, and the presence of its structural analogs including L-tyrosine, L-tryptophan and L-phenylalanine did not show any competitive inhibition. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products in equimolar quantities of the substrate used. The degradation of mimosine into a ring compound, 3H4P with the release of ammonia indicates that MidD of Rhizobium sp. strain TAL1145 is a C-N lyase.