Don Roth

Identification of a plant-encoded analog of PKR, the mammalian double-stranded RNA-dependent protein kinase.

Plant virus or viroid infection stimulates the phosphorylation of a plant-encoded protein of Mr 68,000 to 70,000 (now termed pPKR) that is associated with double-stranded RNA-stimulated protein kinase activity. Using various biochemical and immunological comparisons, we have demonstrated that this plant protein is an analog of the mammalian PKR enzymes. pPKR is both cytosolic and ribosome associated, similar to mammalian PKR, and appears to be capable of phosphorylating exogenous histones. Monoclonal antiserum to the human PKR as well as antiserum to a conserved double-stranded RNA-binding domain present on mammalian PKR demonstrated cross-reactivity with pPKR. Likewise, polyclonal antiserum to the pPKR detected the mouse and human PKR in western blot analysis. Northern blot analysis of a mammalian PKR cDNA detected a specific 2.5-kb transcript present in plant poly(A)+ RNA.

Identification of Non-Pathogenic Xanthomonas Strains Associated with Plants

Summary A total of 70 presumptive non-pathogenic yellow-pigmented strains from different origins were identified to the genus Xanthomonas with the genus-specific monoclonal antibodies (XI and XII), and fatty acid methyl ester (FAME) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein databases. On the basis of FAME profiles and SDS-PAGE protein patterns, these non-pathogenic xanth- omonads formed a heterogeneous population. Fourty-two strains were identified as the same species by both methods, whereas five strains were identified as different species. Only eight strains remained unidentified by both methods, whereas in 15 cases the identification was ambiguous. The identification at pathovar level was usually ambiguous and not concordant, which indicated that the non-pathogenic xanthomonads cannot be classified in the described pathovars. None of the non-pathogenic xanth- omonads was identified as belonging to the pathovars of the plant from which they were isolated.

Specific in vitro phosphorylation of plant eIF2alpha by eukaryotic eIF2alpha kinases.

Phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) is known to be an important translational control mechanism in all eukaryotes with the major exception of plants. Regulation of mammalian and yeast eIF2α activity is directly governed by specific phosphorylation on Ser-51. We now demonstrate that recombinant wheat wild-type (51S) but not mutant 51-Ala (51A) protein is phosphorylated by human PKR and yeast GCN2, which are defined eIF2α kinases. Further, only wheat wild-type eIF2α is a substrate for plant-encoded, double-stranded RNA-dependent kinase (pPKR) activity. Plant PKR and GCN2 phosphorylate recombinant yeast eIF2α 51S but not the 51A mutant demonstrating that pPKR has recognition site capability similar to established eIF2α kinases. A truncated version of wild-type wheat eIF2α containing 51S but not the KGYID motif is not phosphorylated by either hPKR or pPKR suggesting that this putative eIF2α kinase docking domain is essential for phosphorylation. Taken together, these results demonstrate the homology among eukaryotic eIF2α species and eIF2α kinases and support the presence of a plant eIF2α phosphorylation pathway.

Temporal regulation of tobacco mosaic virus-induced phosphorylation of a host encoded protein.

The in vitro and in vivo phosphorylation of a plant encoded protein (p68) associated with dsRNA-dependent protein kinase activity was stimulated at specific time intervals following infection by tobacco mosaic virus or electroporation with dsRNA. The level of p68 phosphorylation in infected and mock inoculated protoplasts did not differ significantly until 6 hr. post-infection, when the basal level of phosphorylation increased 2-3 fold in infected protoplasts. Maximum phosphorylation of p68 occurred between 8-12 hr post-infection and then declined but, at least until 72 hr. post-infection, it was significantly greater than in mock inoculated protoplasts.

Differential localization and accumulation of the plant double stranded RNA-dependent protein kinase during virus infection

Abstract Phosphorylation of the plant encoded, double-stranded (dsRNA)-dependent protein kinase (pPKR) is significantly enhanced over basal levels during early phases of virus and viroid infection. It is unclear, however, if corresponding pPKR protein levels are affected by pathogenesis. We now show that virus infection differentially effects pPKR protein levels. Significantly, cytosolic associated pPKR protein levels are induced 3–5 fold compared to levels in extracts from mock inoculated leaves. However, ribosome-associated pPKR protein levels from virus infected tissues decreased approximately 4-fold relative to levels in extracts from mock inoculated tissues. Further, the level of cytosolic-associated pPKR capable of binding to dsRNA declined during early TMV infection events suggesting that viral dsRNAs bind to pPKR in the cytosol during initial phases of pathogenesis.

An assembly model of Rice dwarf virus particle

The Phytoreovirus rice dwarf virus (RDV) has a complex nucleocapsid architecture composed of multiple proteins and RNAs. However, specific RNA-protein and protein-protein interactions involved in virion packaging have not been entirely elucidated. In order to define mechanisms governing RDV particle assembly, interactions between individual components were analyzed both in vivo and in vitro. The P7 core protein binds specifically and with high affinity to all 12 genomic RDV dsRNAs. P1, a putative RNA polymerase, P5, a putative guanyltransferase and P7 are encapsidated within the virion and also bind viral transcripts based upon in vitro binding assays. P1, P5, P7 and genomic dsRNAs were lacking in empty particles purified from infected tissues that also yielded fractions containing intact, infectious particles. In addition, P7 forms complexes with P1 and P3, a core capsid protein, in viral particles. These results indicate the possibility that core proteins and dsRNAs interact as one unit suggesting a mechanism for assortment of viral RNAs and subsequent packaging into core particles.

A covert authentication and security solution for GMOs

BackgroundProliferation and expansion of security risks necessitates new measures to ensure authenticity and validation of GMOs. Watermarking and other cryptographic methods are available which conceal and recover the original signature, but in the process reveal the authentication information. In many scenarios watermarking and standard cryptographic methods are necessary but not sufficient and new, more advanced, cryptographic protocols are necessary.ResultsHerein, we present a new crypto protocol, that is applicable in broader settings, and embeds the authentication string indistinguishably from a random element in the signature space and the string is verified or denied without disclosing the actual signature. Results show that in a nucleotide string of 1000, the algorithm gives a correlation of 0.98 or higher between the distribution of the codon and that of E. coli, making the signature virtually invisible.ConclusionsThis algorithm may be used to securely authenticate and validate GMOs without disclosing the actual signature. While this protocol uses watermarking, its novelty is in use of more complex cryptographic techniques based on zero knowledge proofs to encode information.

Improving Dependability and Precision of Data Encoding in DNA

DNA storage of information is emerging as the next-generation approach to archiving vast amounts of data. Various sophisticated approaches for data storage in DNA have been proposed. Herein we present a multistep algorithm designed to detect and/or correct errors introduced at any stage of the DNA storage process, including those during message DNA generation, and propose refinements designed to ensure authenticity and correctness of each individual encoded DNA block. In addition, the algorithm allows authentic decoding without a reference sequence or message meaning. The algorithm is designed based on principles underlying provably secure cryptographic systems. Importantly, our new algorithm compares favorably with current ones in terms of ease of implementation and message expansion. In cases where reads are error-free, our algorithm should be faster than current alignment techniques. Without knowing the original data, a certificate is generated that confirms that the obtained data are exactly the same as the original. Our algorithm has applications to DNA steganography, sequence alignment, fast identification of correct reads in next generation sequencing and to message security.