Kranthi K. Mandadi

Plant Immune Responses Against Viruses: How Does a Virus Cause Disease?

Plants respond to pathogens using elaborate networks of genetic interactions. Recently, significant progress has been made in understanding RNA silencing and how viruses counter this apparently ubiquitous antiviral defense. In addition, plants also induce hypersensitive and systemic acquired resistance responses, which together limit the virus to infected cells and impart resistance to the noninfected tissues. Molecular processes such as the ubiquitin proteasome system and DNA methylation are also critical to antiviral defenses. Here, we provide a summary and update of advances in plant antiviral immune responses, beyond RNA silencing mechanisms—advances that went relatively unnoticed in the realm of RNA silencing and nonviral immune responses. We also document the rise of Brachypodium and Setaria species as model grasses to study antiviral responses in Poaceae, aspects that have been relatively understudied, despite grasses being the primary source of our calories, as well as animal feed, forage, recreation, and biofuel needs in the 21st century. Finally, we outline critical gaps, future prospects, and considerations central to studying plant antiviral immunity. To promote an integrated model of plant immunity, we discuss analogous viral and nonviral immune concepts and propose working definitions of viral effectors, effector-triggered immunity, and viral pathogen-triggered immunity.

Development of transcriptomic resources for interrogating the biosynthesis of monoterpene indole alkaloids in medicinal plant species.

The natural diversity of plant metabolism has long been a source for human medicines. One group of plant-derived compounds, the monoterpene indole alkaloids (MIAs), includes well-documented therapeutic agents used in the treatment of cancer (vinblastine, vincristine, camptothecin), hypertension (reserpine, ajmalicine), malaria (quinine), and as analgesics (7-hydroxymitragynine). Our understanding of the biochemical pathways that synthesize these commercially relevant compounds is incomplete due in part to a lack of molecular, genetic, and genomic resources for the identification of the genes involved in these specialized metabolic pathways. To address these limitations, we generated large-scale transcriptome sequence and expression profiles for three species of Asterids that produce medicinally important MIAs: Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina. Using next generation sequencing technology, we sampled the transcriptomes of these species across a diverse set of developmental tissues, and in the case of C. roseus, in cultured cells and roots following elicitor treatment. Through an iterative assembly process, we generated robust transcriptome assemblies for all three species with a substantial number of the assembled transcripts being full or near-full length. The majority of transcripts had a related sequence in either UniRef100, the Arabidopsis thaliana predicted proteome, or the Pfam protein domain database; however, we also identified transcripts that lacked similarity with entries in either database and thereby lack a known function. Representation of known genes within the MIA biosynthetic pathway was robust. As a diverse set of tissues and treatments were surveyed, expression abundances of transcripts in the three species could be estimated to reveal transcripts associated with development and response to elicitor treatment. Together, these transcriptomes and expression abundance matrices provide a rich resource for understanding plant specialized metabolism, and promotes realization of innovative production systems for plant-derived pharmaceuticals.

Novel triterpenoid from Citrus aurantium L. possesses chemopreventive properties against human colon cancer cells

Potential cancer preventive constituents of sour orange (Citrus aurantium L.) were isolated and identified from EtOAc extract of sour orange. Crude EtOAc extract was purified using silica gel column chromatography to isolate two putative bioactive compounds. The purity of the isolated compounds was analyzed by TLC and HPLC. The structures of the two compounds were identified by one-dimensional ((1)H, (13)C) and two-dimensional ((1)H-H and (1)H-(13)C) NMR experiments as isolimonic acid and a novel compound named as ichanexic acid. Stereochemical assignment of the protons for both the compounds was made using one-dimensional nuclear Overhauser enhancement (nOe) experiments. The identified compounds were tested for the inhibition of human colon cancer cells (HT-29) proliferation, apoptosis, and on non-cancerous (COS-1 fibroblast) cells. Cell proliferation, arrest of cell growth, and induction of apoptosis were determined by MTT assay, flow cytometry, and nuclear staining methods, respectively. The MTT assay indicated that both the compounds exhibited differential inhibition at various concentrations. Significant arrest of cell growth by isolimonoic acid was noticed within 24h of treatment on the HT-29 colon cancer cells at a concentration as low as 5.0microM (P=0.005) and by ichanexic acid at 10.0microM (P=0.011). None of the compounds exerted any apparent cytostatic effects on the non-cancerous COS-1 fibroblast cells. Both the compounds exerted nearly 4- to 5-fold increase in the counts of G2/M stage cells at 5microM indicating a potential role in the cell cycle arrest as well as possible lead structures for the development of cancer chemopreventive and therapeutic agents. To the best of our knowledge, this is the first report on isolation, identification of isolimonic acid in its native form, and compound 2 was found to a novel and identified as ichanexic acid.

Genome-Wide Analysis of Alternative Splicing Landscapes Modulated during Plant-Virus Interactions in Brachypodium distachyon

Virus infection altered alternative splicing in >100 Brachypodium distachyon defense-related genes, with little effect on the genome-wide ratios of different splicing types. In eukaryotes, alternative splicing (AS) promotes transcriptome and proteome diversity. The extent of genome-wide AS changes occurring during a plant-microbe interaction is largely unknown. Here, using high-throughput, paired-end RNA sequencing, we generated an isoform-level spliceome map of Brachypodium distachyon infected with Panicum mosaic virus and its satellite virus. Overall, we detected ∼44,443 transcripts in B. distachyon, ∼30% more than those annotated in the reference genome. Expression of ∼28,900 transcripts was ≥2 fragments per kilobase of transcript per million mapped fragments, and ∼42% of multi-exonic genes were alternatively spliced. Comparative analysis of AS patterns in B. distachyon, rice (Oryza sativa), maize (Zea mays), sorghum (Sorghum bicolor), Arabidopsis thaliana, potato (Solanum tuberosum), Medicago truncatula, and poplar (Populus trichocarpa) revealed conserved ratios of the AS types between monocots and dicots. Virus infection quantitatively altered AS events in Brachypodium with little effect on the AS ratios. We discovered AS events for >100 immune-related genes encoding receptor-like kinases, NB-LRR resistance proteins, transcription factors, RNA silencing, and splicing-associated proteins. Cloning and molecular characterization of SCL33, a serine/arginine-rich splicing factor, identified multiple novel intron-retaining splice variants that are developmentally regulated and modulated during virus infection. B. distachyon SCL33 splicing patterns are also strikingly conserved compared with a distant Arabidopsis SCL33 ortholog. This analysis provides new insights into AS landscapes conserved among monocots and dicots and uncovered AS events in plant defense-related genes.

Characterization of a Viral Synergism in the Monocot Brachypodium distachyon Reveals Distinctly Altered Host Molecular Processes Associated with Disease

Panicum mosaic virus (PMV) and its satellite virus (SPMV) together infect several small grain crops, biofuel, and forage and turf grasses. Here, we establish the emerging monocot model Brachypodium (Brachypodium distachyon) as an alternate host to study PMV- and SPMV-host interactions and viral synergism. Infection of Brachypodium with PMV+SPMV induced chlorosis and necrosis of leaves, reduced seed set, caused stunting, and lowered biomass, more than PMV alone. Toward gaining a molecular understanding of PMV- and SPMV-affected host processes, we used a custom-designed microarray and analyzed global changes in gene expression of PMV- and PMV+SPMV-infected plants. PMV infection by itself modulated expression of putative genes functioning in carbon metabolism, photosynthesis, metabolite transport, protein modification, cell wall remodeling, and cell death. Many of these genes were additively altered in a coinfection with PMV+SPMV and correlated to the exacerbated symptoms of PMV+SPMV coinfected plants. PMV+SPMV coinfection also uniquely altered expression of certain genes, including transcription and splicing factors. Among the host defenses commonly affected in PMV and PMV+SPMV coinfections, expression of an antiviral RNA silencing component, SILENCING DEFECTIVE3, was suppressed. Several salicylic acid signaling components, such as pathogenesis-related genes and WRKY transcription factors, were up-regulated. By contrast, several genes in jasmonic acid and ethylene responses were down-regulated. Strikingly, numerous protein kinases, including several classes of receptor-like kinases, were misexpressed. Taken together, our results identified distinctly altered immune responses in monocot antiviral defenses and provide insights into monocot viral synergism.

BT2, a BTB Protein, Mediates Multiple Responses to Nutrients, Stresses, and Hormones in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) gene BT2 encodes a 41-kD protein that possesses an amino-terminal BTB domain, a central TAZ domain, and a carboxyl-terminal calmodulin-binding domain. We previously demonstrated that BT2 could activate telomerase expression in mature Arabidopsis leaves. Here, we report its distinct role in mediating diverse hormone, stress, and metabolic responses. We serendipitously discovered that steady-state expression of BT2 mRNA was regulated diurnally and controlled by the circadian clock, with maximum expression in the dark. This pattern of expression suggested that BT2 mRNA could be linked to the availability of photosynthate in the plant. Exogenous sugars decreased BT2 expression, whereas exogenous nitrogen increased expression. bt2 loss-of-function mutants displayed a hypersensitive response to both sugar-mediated inhibition of germination and abscisic acid (ABA)-mediated inhibition of germination, thus supporting a role of ABA in sugar signaling in germination and development. Moreover, constitutive expression of BT2 imparted resistance to both sugars and ABA at germination, suggesting that BT2 suppresses sugar and ABA responses. In support of the previously described antagonistic relationship between ABA and auxin, we found that BT2 positively regulated certain auxin responses in plants, as revealed by knocking down BT2 expression in the high-auxin mutant yucca. Accumulation of BT2 mRNA was affected by a variety of hormones, nutrients, and stresses, and BT2 was required for responses to many of these same factors. Together, these results suggest that BT2 is a central component of an interconnected signaling network that detects and responds to multiple inputs.

Citrus bioactive compounds improve bone quality and plasma antioxidant activity in orchidectomized rats

BACKGROUND We reported that citrus consumption improves bone quality in orchidectomized male rats. In the present study, effects of feeding citrus bioactive compounds and crude extract on bone quality in orchidectomized rats were evaluated. METHODS Seventy 90-days-old male rats were randomly assigned to five groups for 60 days of feeding study. The treatment groups were SHAM-control, orchidectomy (ORX), ORX+crude extract, ORX+limonin, and ORX+naringin. At termination, animals were euthanized, blood was collected for the plasma antioxidant status. Bone resorption and bone formation markers in the blood and urine were evaluated. Bone quality in the femur and the 5th lumbar and the total calcium concentration in the bones and excreta were evaluated. RESULTS Orchidectomy lowered (p<0.05) plasma antioxidant capacity, bone quality, and bone calcium; elevated (p<0.05) TRAP, deoxypyridinoline (DPD), and calcium excretion; and did not change the plasma IGF-I in comparison to the SHAM group. The citrus crude extract or the purified bioactive compounds increased (p<0.05) the plasma antioxidant status, plasma IGF-I, and bone density, preserved (p<0.05) the concentration of calcium in the femur and in the 5th lumbar, and numerically improved bone strength. The crude extract and the bioactive compounds decreased (p<0.05) fecal excretion of calcium, numerically lowered the urinary excretion of calcium, and suppressed (p<0.05) the plasma TRAP activity without affecting (p>0.1) urinary excretion of DPD in comparison to the ORX group. CONCLUSIONS Potential benefit of the citrus crude extract and its bioactive compounds on bone quality appears to preserve bone calcium concentration and increase antioxidant status.

Regulation of Telomerase in Arabidopsis by BT2, an Apparent Target of TELOMERASE ACTIVATOR1

Telomerase, an enzyme essential for the synthesis and maintenance of telomeric DNA and the long-term stability of the genome, is developmentally regulated in plants. Telomerase activity is abundant in reproductive organs but low or undetectable in vegetative organs. Treatment with exogenous auxin, however, overrides this developmental control and induces telomerase in mature leaves. The Arabidopsis thaliana transcription factor TELOMERASE ACTIVATOR1 (TAC1) potentiates some responses to auxin, including the induction of telomerase activity in leaves. Here, we report that BT2, a protein with BTB, TAZ, and calmodulin binding domains, is an essential component of the TAC1-mediated telomerase activation pathway. Steady state concentration of BT2 mRNA increases in response to TAC1 expression, and TAC1 specifically binds the BT2 promoter both in vitro and in yeast one-hybrid assays. Constitutive expression of BT2 induces telomerase activity in leaves, whereas a null mutation of BT2 blocks TAC1-mediated telomerase induction, indicating that BT2 acts downstream of TAC1 to regulate telomerase activity in mature vegetative organs.

Red Mexican Grapefruit: A Novel Source for Bioactive Limonoids and their Antioxidant Activity

Abstract Citrus limonoids have shown to inhibit the growth of cancer in colon, lung, mouth, stomach and breast in animal and cell culture studies. For the first time in the present study, an attempt has been made to isolate antioxidant fractions and five limonoids from red Mexican grapefruit seeds. Defatted seed powder was successively extracted with hexane, ethyl acetate (EtOAc), acetone, methanol (MeOH) and MeOH/water and the extracts were concentrated under vacuum. Radical scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and total phenolic content were also measured for comparison with the antioxidant capacity in the phosphomolybdenum method for the above extracts. Acetone and MeOH extracts, respectively, showed the highest (85.7%) and lowest (53.3%) radical scavenging activity, at 500 ppm. The total phenolic contents were found to be highest in the acetone extract (15.94%) followed by the MeOH extract (5.92%), ethyl acetate extract (5.54%) and water extract (5.26%). Antioxidant capacity of the extracts as equivalents to ascorbic acid (μmol/g of the extract) was in the order, EtOAc extract > acetone extract > water extract > methanol extract. Furthermore, the EtOAC and acetone extracts were loaded onto silica gel columns to obtain four limonoid aglycons. MeOH fraction was loaded onto a dowex-50 and sepabeads resin column to obtain a limonoid glucoside. The purity of the isolated five compounds was analyzed by HPLC using a C18 column and UV detection at 210 nm. Finally, the structures of the compounds were identified as obacunone, nomilin, limonin, deacetylnomilin (DAN) and limonin-17-β-d-glucopyranoside (LG) using 1H and 13C NMR studies.

Comparative Analysis of Antiviral Responses in Brachypodium distachyon and Setaria viridis Reveals Conserved and Unique Outcomes Among C3 and C4 Plant Defenses

Viral diseases cause significant losses in global agricultural production, yet little is known about grass antiviral defense mechanisms. We previously reported on host immune responses triggered by Panicum mosaic virus (PMV) and its satellite virus (SPMV) in the model C3 grass Brachypodium distachyon. To aid comparative analyses of C3 and C4 grass antiviral defenses, here, we establish B. distachyon and Setaria viridis (a C4 grass) as compatible hosts for seven grass-infecting viruses, including PMV and SPMV, Brome mosaic virus, Barley stripe mosaic virus, Maize mild mottle virus, Sorghum yellow banding virus, Wheat streak mosaic virus (WSMV), and Foxtail mosaic virus (FoMV). Etiological and molecular characterization of the fourteen grass-virus pathosystems showed evidence for conserved crosstalk among salicylic acid (SA), jasmonic acid, and ethylene pathways in B. distachyon and S. viridis. Strikingly, expression of PHYTOALEXIN DEFICIENT4, an upstream modulator of SA signaling, was consistently suppressed during most virus infections in B. distachyon and S. viridis. Hierarchical clustering analyses further identified unique antiviral responses triggered by two morphologically similar viruses, FoMV and WSMV, and uncovered other host-dependent effects. Together, the results of this study establish B. distachyon and S. viridis as models for the analysis of plant-virus interactions and provide the first framework for conserved and unique features of C3 and C4 grass antiviral defenses.