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Katalin A. Hudak - One of the best experts on this subject based on the ideXlab platform.

  • Image_1_Integration of the Pokeweed miRNA and mRNA Transcriptomes Reveals Targeting of Jasmonic Acid-Responsive Genes.PDF
    2018
    Co-Authors: Kira Catherine Maxine Neller, Alexander Klenov, Juan C. Guzman, Katalin A. Hudak
    Abstract:

    The American Pokeweed plant, Phytolacca americana, displays broad-spectrum resistance to plant viruses and is a heavy metal hyperaccumulator. However, little is known about the regulation of biotic and abiotic stress responses in this non-model plant. To investigate the control of miRNAs in gene expression, we sequenced the small RNA transcriptome of Pokeweed treated with jasmonic acid (JA), a hormone that mediates pathogen defense and stress tolerance. We predicted 145 miRNAs responsive to JA, most of which were unique to Pokeweed. These miRNAs were low in abundance and condition-specific, with discrete expression change. Integration of paired mRNA-Seq expression data enabled us to identify correlated, novel JA-responsive targets that mediate hormone biosynthesis, signal transduction, and pathogen defense. The expression of approximately half the pairs was positively correlated, an uncommon finding that we functionally validated by mRNA cleavage. Importantly, we report that a Pokeweed-specific miRNA targets the transcript of OPR3, novel evidence that a miRNA regulates a JA biosynthesis enzyme. This first large-scale small RNA study of a Phytolaccaceae family member shows that miRNA-mediated control is a significant component of the JA response, associated with widespread changes in expression of genes required for stress adaptation.

  • Table_3_Integration of the Pokeweed miRNA and mRNA Transcriptomes Reveals Targeting of Jasmonic Acid-Responsive Genes.XLSX
    2018
    Co-Authors: Kira Catherine Maxine Neller, Alexander Klenov, Juan C. Guzman, Katalin A. Hudak
    Abstract:

    The American Pokeweed plant, Phytolacca americana, displays broad-spectrum resistance to plant viruses and is a heavy metal hyperaccumulator. However, little is known about the regulation of biotic and abiotic stress responses in this non-model plant. To investigate the control of miRNAs in gene expression, we sequenced the small RNA transcriptome of Pokeweed treated with jasmonic acid (JA), a hormone that mediates pathogen defense and stress tolerance. We predicted 145 miRNAs responsive to JA, most of which were unique to Pokeweed. These miRNAs were low in abundance and condition-specific, with discrete expression change. Integration of paired mRNA-Seq expression data enabled us to identify correlated, novel JA-responsive targets that mediate hormone biosynthesis, signal transduction, and pathogen defense. The expression of approximately half the pairs was positively correlated, an uncommon finding that we functionally validated by mRNA cleavage. Importantly, we report that a Pokeweed-specific miRNA targets the transcript of OPR3, novel evidence that a miRNA regulates a JA biosynthesis enzyme. This first large-scale small RNA study of a Phytolaccaceae family member shows that miRNA-mediated control is a significant component of the JA response, associated with widespread changes in expression of genes required for stress adaptation.

  • homodimerization of Pokeweed antiviral protein as a mechanism to limit depurination of Pokeweed ribosomes
    Molecular Plant Pathology, 2010
    Co-Authors: Marina E Tourlakis, Rajita A Karran, Leroi V Desouza, Katalin A. Hudak
    Abstract:

    SUMMARY Ribosome inactivating proteins are glycosidases synthesized by many plants and have been hypothesized to serve in defence against pathogens. These enzymes catalytically remove a conserved purine from the sarcin/ricin loop of the large ribosomal RNA, which has been shown in vitro to limit protein synthesis. The resulting toxicity suggests that plants may possess a mechanism to protect their ribosomes from depurination during the synthesis of these enzymes. For example, Pokeweed antiviral protein (PAP) is cotranslationally inserted into the lumen of the endoplasmic reticulum and travels via the endomembrane system to be stored in the cell wall. However, some PAP may retrotranslocate across the endoplasmic reticulum membrane to be released back into the cytosol, thereby exposing ribosomes to depurination. In this work, we isolated and characterized a complexed form of the enzyme that exhibits substantially reduced activity. We showed that this complex is a homodimer of PAP and that dimerization involves a peptide that contains a conserved aromatic amino acid, tyrosine 123, located in the active site of the enzyme. Bimolecular fluorescence complementation demonstrated that the homodimer may form in vivo and that dimerization is prevented by the substitution of tyrosine 123 for alanine. The homodimer is a minor form of PAP, observed only in the cytosol of cells and not in the apoplast. Taken together, these data support a novel mechanism for the limitation of depurination of autologous ribosomes by molecules of the protein that escape transport to the cell wall by the endomembrane system.

  • Pokeweed antiviral protein depurinates the sarcin ricin loop of the rrna prior to binding of aminoacyl trna to the ribosomal a site
    RNA, 2006
    Co-Authors: Sheila Mansouri, Emad Nourollahzadeh, Katalin A. Hudak
    Abstract:

    Ribosome-inactivating proteins, such as the Pokeweed antiviral protein (PAP), inhibit translation by depurinating the conserved sarcin/ricin loop of the large ribosomal RNA. Depurinated ribosomes are unable to bind elongation factor 2, and, thus, the translocation step of the elongation cycle is inhibited. Though the consequences of depurination are well characterized, the ribosome conformation required for depurination to take place has not been described. In this report, we correlate biochemical and genetic data to conclude that Pokeweed antiviral protein depurinates the sarcin/ricin loop when the A-site of the ribosomal peptidyl-transferase center is unoccupied. We show that prior incubation of ribosomes with puromycin, an analog of the 3′-terminus of aminoacyl-tRNA, inhibits both binding and depurination by PAP in a concentration-dependent manner. Expression of PAP in the yeast strain mak8-1 results in little depurination unless the cells are lysed, a process that would promote loss of aminoacyl-tRNA from the ribosome. The mak8-1 strain is known to exhibit a higher affinity for aminoacyl-tRNA compared with wild-type cells, and therefore, its ribosomes are more resistant to PAP in vivo. These data contribute to the mechanism of action of Pokeweed antiviral protein; specifically, they have uncovered the ribosomal conformation required for depurination that leads to subsequent translation inhibition.

  • Pokeweed antiviral protein inhibits brome mosaic virus replication in plant cells
    Journal of Biological Chemistry, 2005
    Co-Authors: Daniel Picard, Cheng C Kao, Katalin A. Hudak
    Abstract:

    Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein isolated from the Pokeweed plant (Phytolacca americana) that inhibits the proliferation of several plant and animal viruses. We have shown previously that PAP and nontoxic mutants of PAP can directly depurinate brome mosaic virus (BMV) RNA in vitro, resulting in reduced viral protein translation. Here we expand on these initial studies and, using a barley protoplast system, demonstrate that recombinant PAP and nontoxic mutants isolated from E. coli are able to reduce the accumulation of BMV RNAs in vivo. Pretreatment of only BMV RNA3 with PAP prior to transfection of barley protoplasts reduced the accumulation of all BMV RNAs, with a more severe effect on subgenomic RNA4 levels. Using in vitro RNA synthesis assays, we show that a depurinated template causes the BMV replicase to stall at the template nucleotide adjacent to the missing base. These results provide new insight into the antiviral mechanism of PAP, namely that PAP depurination of BMV RNA impedes both RNA replication and subgenomic RNA transcription. These novel activities are distinct from the PAP-induced reduction of viral RNA translation and represent new targets for the inhibition of viral infection.

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  • Ovipositional preference, fidelity, and fitness of Drosophila suzukii in a co-occurring crop and non-crop host system
    Journal of Pest Science, 2016
    Co-Authors: Lauren M. Diepenbrock, Katharine A. Swoboda-bhattarai, Hannah J. Burrack
    Abstract:

    Drosophila suzukii (Matsumura) is a devastating pest of soft-skinned fruits including blackberries and raspberries. Management of this pest is focused on preventing infestation in crops, but non-crop hosts may play an important role in enabling D. suzukii to persist in the absence of cultivated hosts. Drosophila suzukii may also infest fruits of both crop and non-crop hosts concurrently. Our goals were to determine whether (1) D. suzukii prefers to oviposit in cultivated blackberry, Rubus L. subgenus rubus Watson, or American Pokeweed, Phytolacca americana, a non-crop host commonly found along field edges, (2) D. suzukii prefers to oviposit into the same host from which it emerged, and (3) performance differs between D. suzukii progeny that develop in blackberries or Pokeweed berries. Although the pest was able to infest both hosts at the same rate, we found that D. suzukii females emerging from Pokeweed preferred to oviposit into blackberries, while females emerging from blackberry had no preference. Progeny that developed in blackberries were more fit than progeny that developed in Pokeweed berries based on several measures. In field locations, cultivated blackberries and Pokeweed berries only overlapped in availability for a short period of time, and infestation rates were variable between blackberries and Pokeweed berries collected during that period. Nonetheless, these results suggest that non-crop hosts may facilitate the invasion of D. suzukii and perpetuate infestation of cultivated hosts under certain circumstances.