Medicago Sativa Defensin1 as a tumor sensitizer for improving chemotherapy: translation from anti-fungal agent to a potential anti-cancer agent.

Plant defensins including Medicago Sativa defensin 1 (MsDef1) are cysteine-rich antifungal peptides which are known for potent broad-spectrum antifungal activity against bacterial or fungal pathogens of plants. The antimicrobial activities of these cationic defensins are attributed to their capacity to bind to cell membranes to create potentially structural defects tin the cell membranes to interact with intracellular target (s) and mediates cytotoxic effects. Our earlier work identified Glucosylceramide (GlcCer) of fungus F. graminearum as a potential target for biological activity. Multi-drug resistant (MDR) cancer cells overexpress GlcCer on the surface of plasma membrane. Hence, MsDef1 may have a potential to bind to GlcCer of MDR cancer cells to induce cell death. We have characterized the three-dimensional structure of MsDef1 and the solution dynamics using of 15N-labeled MsDef1 nuclear magnetic resonance (NMR) spectroscopy which showed that GlcCer binds MsDef1 at two specific sites on the peptide molecule. The ability of MsDef1 to permeate MDR cancer cells was demonstrated by measuring the release of apoptotic ceramide in drug resistant MCF-7R cells. It was also shown that MsDef1 activated dual cell death pathways ceramide and Apoptosis Stimulating Kinase ASK1 by disintegrating GlcCer and oxidizing tumor specific biomarker thioredoxin (Trx) respectively. As a result, MsDef1 sensitizes MDR cancer cells to evoke a better response from Doxorubicin, a front-line chemotherapy for triple negative breast cancer (TNBC) treatment. The combination of MsDef1 and Doxorubicin induced 5 to10-fold greater apoptosis in vitro MDR cells MDA-MB-231R compared to either MsDef1 or Doxorubicin alone. Confocal microscopy revealed that MsDef1 facilitates a) influx of Doxorubicin in MDR cancer cells, b) preferential uptake by MDR cells but not by normal fibroblasts and breast epithelial cells (MCF-10A). These results suggest that MsDef1 targets MDR cancer cells and may find utility as a neoadjuvant chemotherapy. Hence, the extension of antifungal properties of MsDef1 to cancer my result in addressing the MDR problems in cancer.

Biomolecular characterisation of marine microalga in comparison to fishmeal and soymeal as an alternative feed ingredient.

INTRODUCTION: Marine microalgae protein has better solubility and digestibility than other protein-based feeds. Apart from protein, high-value biomolecules have an immense potential to enhance the quality of feed, but knowledge about them is scarce. OBJECTIVE: Marine microalga Picochlorum sp. biomass molecular characterisation along with commonly used protein feed such as fishmeal and soymeal for potential feed ingredients. METHODOLOGY: Liquid chromatography coupled with mass spectrometry (LC-MS) was used for biomolecular characterisation. The correlation of biomolecules sets was evaluated using principal component analysis (PCA) and heatmap clustering. RESULTS: LC-MS identified 116 biomolecules cumulatively among microalga, fishmeal, and soymeal that includes fatty acids, acylglycerols, vitamins, sterols, pigments, nucleotides, unique amino acids, amines, sugars and miscellaneous. These 116 biomolecules were screened based on their functional importance as feed ingredients. Among the different sets of biomolecules, microalga contained a more diverse set of fatty acids, pigments, sterols, and vitamins than acylglycerols, unique amino acids, nucleotides, and sugars. Fishmeal contained a more diverse set of acylglycerols, unique amino acids, nucleotides, and amines, while soymeal contained the highest number of sugars and miscellaneous biomolecules. The PCA confirmed the significance level (P > 95%) and heatmap clustering showed the diversity and relatedness of biomolecules among the microalga, fishmeal, and soymeal. CONCLUSION: This study showed that the marine microalga Picochlorum sp. biomass has a rich source of biomolecules and could complement fishmeal or soymeal in feed and is also sustainable and economical as compared to fishmeal and soymeal.

Microalgae as feed ingredients: recent developments on their role in immunomodulation and gut microbiota of aquaculture species.

Microalgae are rapidly evolving alternative ingredients in food and feed. Desirable nutritional and functional qualities make them high potential sources of feed ingredients. Certain microalgae species are known to accumulate large amounts of protein, containing all essential amino acids while some species contain essential fatty acids and bioactive compounds hence offering several possible health benefits. However, successful inclusion of microalgae-based products in feed requires a clear understanding of physiological responses and microbiota of animals receiving microalgae diets. In this review, key microalgae-based feed ingredients and their effect on gut microbiome and immunomodulatory responses of microalgae fed animals, with a focus on aquatic species will be discussed.

Structural and functional studies of a phosphatidic acid-binding antifungal plant defensin MtDef4: identification of an RGFRRR motif governing fungal cell entry.

MtDef4 is a 47-amino acid cysteine-rich evolutionary conserved defensin from a model legume Medicago truncatula. It is an apoplast-localized plant defense protein that inhibits the growth of the ascomycetous fungal pathogen Fusarium graminearum in vitro at micromolar concentrations. Little is known about the mechanisms by which MtDef4 mediates its antifungal activity. In this study, we show that MtDef4 rapidly permeabilizes fungal plasma membrane and is internalized by the fungal cells where it accumulates in the cytoplasm. Furthermore, analysis of the structure of MtDef4 reveals the presence of a positively charged γ-core motif composed of ß2 and ß3 strands connected by a positively charged RGFRRR loop. Replacement of the RGFRRR sequence with AAAARR or RGFRAA abolishes the ability of MtDef4 to enter fungal cells, suggesting that the RGFRRR loop is a translocation signal required for the internalization of the protein. MtDef4 binds to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Amino acid substitutions in the RGFRRR sequence which abolish the ability of MtDef4 to enter fungal cells also impair its ability to bind PA. These findings suggest that MtDef4 is a novel antifungal plant defensin capable of entering into fungal cells and affecting intracellular targets and that these processes are mediated by the highly conserved cationic RGFRRR loop via its interaction with PA.

Structure-activity determinants in antifungal plant defensins MsDef1 and MtDef4 with different modes of action against Fusarium graminearum.

Plant defensins are small cysteine-rich antimicrobial proteins. Their three-dimensional structures are similar in that they consist of an α-helix and three anti-parallel ß-strands stabilized by four disulfide bonds. Plant defensins MsDef1 and MtDef4 are potent inhibitors of the growth of several filamentous fungi including Fusarium graminearum. However, they differ markedly in their antifungal properties as well as modes of antifungal action. MsDef1 induces prolific hyperbranching of fungal hyphae, whereas MtDef4 does not. Both defensins contain a highly conserved γ-core motif (GXCX(3-9)C), a hallmark signature present in the disulfide-stabilized antimicrobial peptides, composed of ß2 and ß3 strands and the interposed loop. The γ-core motifs of these two defensins differ significantly in their primary amino acid sequences and in their net charge. In this study, we have found that the major determinants of the antifungal activity and morphogenicity of these defensins reside in their γ-core motifs. The MsDef1-γ4 variant in which the γ-core motif of MsDef1 was replaced by that of MtDef4 was almost as potent as MtDef4 and also failed to induce hyperbranching of fungal hyphae. Importantly, the γ-core motif of MtDef4 alone was capable of inhibiting fungal growth, but that of MsDef1 was not. The analysis of synthetic γ-core variants of MtDef4 indicated that the cationic and hydrophobic amino acids were important for antifungal activity. Both MsDef1 and MtDef4 induced plasma membrane permeabilization; however, kinetic studies revealed that MtDef4 was more efficient in permeabilizing fungal plasma membrane than MsDef1. Furthermore, the in vitro antifungal activity of MsDef1, MsDef1-γ4, MtDef4 and peptides derived from the γ-core motif of each defensin was not solely dependent on their ability to permeabilize the fungal plasma membrane. The data reported here indicate that the γ-core motif defines the unique antifungal properties of each defensin and may facilitate de novo design of more potent antifungal peptides.

Requirement of the galU gene for polysaccharide production by and pathogenicity and growth In Planta of Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri is the causal agent of citrus canker, which has a significant impact on citrus production. In this study, we characterized the galU gene of X. citri subsp. citri. Two galU mutants (F6 and D12) were identified in an X. citri subsp. citri EZ-Tn5 Tnp transposon library. Rescue cloning, sequence analysis, and Southern blot analysis indicated that both of these mutants had a single copy of the EZ-Tn5 transposon inserted in galU in the chromosome. Further study showed that galU was required for biosynthesis of extracellular polysaccharides (EPS; xanthan gum) and capsular polysaccharide (CPS) and biofilm formation. Mutation of galU resulted in a loss of pathogenicity for grapefruit. The loss of pathogenicity of a galU mutant resulted from its inability to grow in planta rather than from the effect on virulence genes. Quantitative reverse transcription-PCR assays indicated that mutation of galU did not impair the expression of key virulence genes, such as pthA of X. citri subsp. citri. Although D12 had a growth rate similar to that of the wild-type strain in nutrient broth, no D12 population became established in the intercellular spaces of citrus leaves. Coinoculation of a galU mutant with the wild-type strain did not promote growth of the galU mutant in planta. Defects in EPS and CPS production, pathogenicity, and growth in planta of the galU mutant were complemented to the wild-type level using plasmid pCGU2.1 containing an intact galU gene. These data indicate that the galU gene contributes to X. citri subsp. citri growth in intercellular spaces and is involved in EPS and CPS synthesis and biofilm formation.

Bacterial diversity analysis of Huanglongbing pathogen-infected citrus, using PhyloChip arrays and 16S rRNA gene clone library sequencing.

The bacterial diversity associated with citrus leaf midribs was characterized for citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rRNA gene microarrays and 16S rRNA gene clone library sequencing to determine the microbial community composition for symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria in 15 phyla were present in the citrus leaf midribs, while 20 orders in 8 phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs than in asymptomatic midribs. "Candidatus Liberibacter asiaticus" was detected at a very low level in asymptomatic plants but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis results were further verified by sequencing 16S rRNA gene clone libraries, which indicated the dominance of "Candidatus Liberibacter asiaticus" in symptomatic leaves. These data implicate "Candidatus Liberibacter asiaticus" as the pathogen responsible for HLB disease.

Response of sweet orange (Citrus sinensis) to 'Candidatus Liberibacter asiaticus' infection: microscopy and microarray analyses.

Citrus greening or huanglongbing (HLB) is a devastating disease of citrus. HLB is associated with the phloem-limited fastidious prokaryotic alpha-proteobacterium 'Candidatus Liberibacter spp.' In this report, we used sweet orange (Citrus sinensis) leaf tissue infected with 'Ca. Liberibacter asiaticus' and compared this with healthy controls. Investigation of the host response was examined with citrus microarray hybridization based on 33,879 expressed sequence tag sequences from several citrus species and hybrids. The microarray analysis indicated that HLB infection significantly affected expression of 624 genes whose encoded proteins were categorized according to function. The categories included genes associated with sugar metabolism, plant defense, phytohormone, and cell wall metabolism, as well as 14 other gene categories. The anatomical analyses indicated that HLB bacterium infection caused phloem disruption, sucrose accumulation, and plugged sieve pores. The up-regulation of three key starch biosynthetic genes including ADP-glucose pyrophosphorylase, starch synthase, granule-bound starch synthase and starch debranching enzyme likely contributed to accumulation of starch in HLB-affected leaves. The HLB-associated phloem blockage resulted from the plugged sieve pores rather than the HLB bacterial aggregates since 'Ca. Liberibacter asiaticus' does not form aggregate in citrus. The up-regulation of pp2 gene is related to callose deposition to plug the sieve pores in HLB-affected plants.

In planta distribution of 'Candidatus Liberibacter asiaticus' as revealed by polymerase chain reaction (PCR) and real-time PCR.

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide, and is caused by a phloem-limited fastidious prokaryotic alpha-proteobacterium that is yet to be cultured. In this study, a combination of traditional polymerase chain reaction (PCR) and real-time PCR targeting the putative DNA polymerase and 16S rDNA sequence of 'Candidatus Liberibacter asiaticus,' respectively, were used to examine the distribution and movement of the HLB pathogen in the infected citrus tree. We found that 'Ca. Liberibacter asiaticus' was distributed in bark tissue, leaf midrib, roots, and different floral and fruit parts, but not in endosperm and embryo, of infected citrus trees. Quantification analysis of the HLB bacterium indicated that it was distributed unevenly in planta and ranged from 14 to 137,031 cells/mug of total DNA in different tissues. A relatively high concentration of 'Ca. Liberibacter asiaticus' was observed in fruit peduncles. Our data from greenhouse-infected plants also indicated that 'Ca. Liberibacter asiaticus' was transmitted systemically from infection site to different parts of the plant. Understanding the distribution and movement of the HLB bacterium inside an individual citrus tree is critical for discerning its virulence mechanism and to develop management strategies for HLB.

Fusarium verticillioides GAP1, a gene encoding a putative glycolipid-anchored surface protein, participates in conidiation and cell wall structure but not virulence.

Fusarium verticillioides is an important pathogen of maize that causes ear rot and produces the mycotoxins known as fumonisins. To date, knowledge of pathogenicity and the regulation of fumonisin biosynthesis in F. verticillioides is limited. Here, the molecular characterization of GAP1, a gene encoding a putative 540 aa protein that belongs to a glycolipid-anchored surface (GAS) protein family, is presented. F. verticillioides GAP1 was identified as an expressed sequence tag (EST) upregulated in a culture condition conducive to conidiation and fumonisin B(1) (FB(1)) production. GAP1 null mutants GAM126 (Deltagap1 : : HYG) and GAG8 (Deltagap1 : : GEN) exhibited restricted growth, with more aerial hyphae than their wild-type progenitor on solid media. No defect in mycelial mass or filamentous growth was observed when the GAM126 and GAG8 strains were grown in liquid media under shaking conditions. When grown in suspended conditions, GAM126 and GAG8 strains produced significantly fewer conidia and produced comparatively densely branched hyphae. Concanavalin A staining indicated that the GAP1 deletion altered the cell wall carbohydrate composition/deposition process. Deletion of GAP1 did not affect the production level of FB(1) or F. verticillioides virulence on maize seedlings and stalks. Complementation of GAM126 with the wild-type GAP1 gene restored growth, conidiation and cell wall abnormality phenotypes. The results suggest that GAP1 is associated with growth, development and conidiation in F. verticillioides, but not with pathogenicity or regulation of FB(1).

Fusarium verticillioides GBB1, a gene encoding heterotrimeric G protein beta subunit, is associated with fumonisin B biosynthesis and hyphal development but not with fungal virulence.

SUMMARY: Fusarium verticillioides (Sacc.) Nirenberg (teleomorph Gibberella moniliformis Wineland) is a maize pathogen that causes ear rots and stalk rots. The fungus also produces a group of mycotoxins, fumonisins, on infected ears, which cause considerable health and economic concerns for humans and animals worldwide. To date, our understanding of the molecular mechanisms associated with fungal virulence and fumonisin biosynthesis in F. verticillioides is limited. In this study, GBB1, a gene encoding a putative beta subunit of a heterotrimeric G protein, was disrupted and the effects on fumonisin biosynthesis and virulence were evaluated. A GBB1 deletion mutant (Deltagbb1) showed no significant differences in radial growth and mycelial mass but produced significantly less fumonisin B(1 )(FB(1)) than its wild-type progenitor. HPLC analysis showed that Deltagbb1 produced less than 10 p.p.m. FB(1) while the wild-type produced over 140 p.p.m. when strains were grown on cracked corn kernels. Reduced expression of the key FB(1 )biosynthetic genes, FUM1 and FUM8, in Deltagbb1 provides further evidence that GBB1 is involved in FB(1) regulation. Stalk rot virulence, as measured by mean lesion length and by area, was not significantly different in Deltagbb1 compared with the wild-type, suggesting that GBB1 does not regulate virulence in F. verticillioides. Developmentally, hyphae of Deltagbb1 do not deviate from the original axis of polarity established upon germ tube emergence in contrast to wild-type hyphae that meander on and off axis as they grow. Complementation of Deltagbb1 with GBB1 restored FB(1) production and hyphal growth to wild-type. The results of this study demonstrate that heterotrimeric G protein beta subunit plays an important role in regulation of FB(1) biosynthesis and hyphal growth, but not virulence in F. verticillioides.

FSR1 is essential for virulence and female fertility in Fusarium verticillioides and F. graminearum.

Fusarium verticillioides (teleomorph Gibberella moniliformis) and F. graminearum (teleomorph G. zeae) are well known to cause devastating diseases on cereal crops. Despite their importance, our understanding of the molecular mechanisms involved in these host-pathogen interactions is limited. The FSR1 locus in F. verticillioides was identified by screening REMI mutants for loss of virulence in maize stalk rot inoculation studies. FSR1 encodes an 823-codon open reading frame interrupted by two introns. The Fsr1 protein shares 60% sequence identity with the Sordaria macrospora Pro11, a multimodular protein with four putative protein-protein binding domains (caveolin-binding domain, coiled-coil structure, calmodulin-binding motif, and seven-WD40 repeats), which plays a regulatory role in cell differentiation and ascocarp development. Our data demonstrate that FSR1 is essential for female fertility and virulence in F. verticillioides. Significantly, targeted disruption of the FSR1 ortholog in F. graminearum (FgFSR1) reduced virulence on barley and deterred perithecia formation. Cross-complementation experiments demonstrated that the gene function is conserved in the two Fusarium species. FSR1 is expressed constitutively, and we hypothesize that Fsr1 regulates virulence by acting as a scaffold for a signal transduction pathway. A survey of available genome databases indicates Fsr1 homologs are present in a number of filamentous fungi and animal systems but not in budding yeast or plants. A maximum likelihood analysis of this gene family reveals well-supported monophyletic clades associated with fungi and animals.

The putative monomeric G-protein GBP1 is negatively associated with fumonisin B production in Fusarium verticillioides.

SUMMARY Fumonisin B(1) (FB(1)) is a mycotoxin produced by Fusarium verticillioides that contaminates maize. FB(1) has been linked to a number of human and animal mycotoxicoses worldwide. Despite its significance, our understanding of the FB(1) biosynthesis regulatory mechanisms is limited. Here, we describe F. verticillioides GBP1, encoding a monomeric G-protein, and its role in FB(1) biosynthesis. GBP1 was discovered as an expressed sequence tag (EST) up-regulated in the F. verticillioides fcc1 mutant that showed reduced conidiation and no FB(1) biosynthesis when grown on maize kernels. Sequence analysis showed that GBP1 encodes a putative 368-amino-acid protein with similarity to DRG and Obg subclasses of G-proteins that are involved in development and stress responses. A GBP1 knockout mutant (Deltagbp1) exhibited normal growth, but increased FB(1) production (> 58%) compared with the wild-type when grown on corn kernels. Complementation of Deltagbp1 with the wild-type GBP1 gene restored FB(1) production levels to that of the wild-type. Our data indicate that GBP1 is negatively associated with FB(1) biosynthesis but not with conidiation in F. verticillioides. The deletion of GBP1 led to up-regulation of key FB(1) biosynthetic genes, FUM1 and FUM8, suggesting that the increased FB(1) production in Deltagbp1 is due to over-expression of FUM genes.