Molecular and Metagenomic Analyses Reveal High Prevalence and Complexity of Viral Infections in French-American Hybrids and North American Grapes.

French-American hybrids and North American grape species play a significant role in Canada's grape and wine industry. Unfortunately, the occurrence of viruses and viral diseases among these locally important non- grapes remains understudied. We report here the results from a large-scale survey to assess the prevalence of 14 viruses among 533 composite samples representing 2665 vines from seven French-American hybrid wine grape cultivars, two North American juice grape cultivars (Concord and Niagara), and the table grape cultivar Sovereign coronation.

Concerning the Etiology of Syrah Decline: A Fresh Perspective on an Old and Complex Issue Facing the Global Grape and Wine Industry.

Syrah decline, first identified in Southern France in the 1990s, has become a major concern in the global grape and wine industry. This disease mainly affects Syrah (Shiraz) grapevines. Characteristic symptoms include the bright and uniform reddening of leaves throughout the canopy in late summer or early fall; the appearance of abnormalities on the trunk, mainly at the graft union (swelling, pits, grooves, and necrosis); and a reduction in vine vigor, yield and berry quality.

Characterizing the Virome of Apple Orchards Affected by Rapid Decline in the Okanagan and Similkameen Valleys of British Columbia (Canada).

Rapid apple decline disease (RAD) has been affecting orchards in the USA and Canada. Although the primary cause for RAD remains unknown, viruses may contribute to the incidence or severity of the disease. We examined the diversity and prevalence of viruses in orchards affected by RAD in the Okanagan and Similkameen Valleys (British Columbia, Canada).

Structures of plasmepsin X from Plasmodium falciparum reveal a novel inactivation mechanism of the zymogen and molecular basis for binding of inhibitors in mature enzyme.

Plasmodium falciparum plasmepsin X (PfPMX), involved in the invasion and egress of this deadliest malarial parasite, is essential for its survival and hence considered as an important drug target. We report the first crystal structure of PfPMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme is involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to PfPMX.

Proteolytic Processing of Plant Proteins by Potyvirus NIa Proteases.

The NIa protease of potyviruses is a chymotrypsin-like cysteine protease related to the picornavirus 3C protease. It is also a multifunctional protein known to play multiple roles during virus infection. Picornavirus 3C proteases cleave hundreds of host proteins to facilitate virus infection.

Activation mechanism of plasmepsins, pepsin-like aspartic proteases from Plasmodium, follows a unique trans-activation pathway.

Plasmodium parasites that cause malaria produce plasmepsins (PMs), pepsin-like aspartic proteases that are important antimalarial drug targets due to their role in host hemoglobin degradation. The enzymes are synthesized as inactive zymogens (pro-PMs), and the mechanism of their conversion to the active, mature forms has not been clearly elucidated. Our structural investigations of vacuolar pro-PMs with truncated prosegment (pro-tPMs) reveal that the formation of the S-shaped dimer is their innate property.

Metagenomic Analysis of Riesling Grapevine Reveals a Complex Virome Including Two New and Divergent Variants of .

The virome of a major white wine grape of cultivar Riesling showing decline and leafroll disease symptoms was analyzed through high-throughput sequencing (HTS) using total RNAs as templates and the Illumina HiSeq 2500 platform. Analysis of HTS data revealed the presence of five viruses and three viroids in the infected vine. These viruses are (GLRaV-1) and GLRaV-3 (genus , family ) and three viruses of the family (namely, [GVA], , and [GRSPaV]).

Characterization of grapevine leafroll-associated virus 3 genetic variants and application towards RT-qPCR assay design.

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widely prevalent and economically important of the complex of RNA viruses associated with grapevine leafroll disease (GLD). Phylogenetic studies have grouped GLRaV-3 isolates into nine different monophyletic groups and four supergroups, making GLRaV-3 a genetically highly diverse virus species. In addition, new divergent variants have been discovered recently around the world.

Survey for major viruses in commercial Vitis vinifera wine grapes in Ontario.

Background: In recent years, the Ontario grape and wine industry has experienced outbreaks of viral diseases across the province. Little is known about the prevalence of viruses and viral diseases in Ontario. Since 2015, we have conducted large-scale surveys for major viruses in commercial wine grapes in order to obtain a comprehensive understanding of the prevalence and severity of viral diseases in Ontario.

Deciphering the mechanism of potent peptidomimetic inhibitors targeting plasmepsins - biochemical and structural insights.

Malaria is a deadly disease killing worldwide hundreds of thousands people each year and the responsible parasite has acquired resistance to the available drug combinations. The four vacuolar plasmepsins (PMs) in Plasmodium falciparum involved in hemoglobin (Hb) catabolism represent promising targets to combat drug resistance. High antimalarial activities can be achieved by developing a single drug that would simultaneously target all the vacuolar PMs.

Understanding the structural basis of substrate recognition by Plasmodium falciparum plasmepsin V to aid in the design of potent inhibitors.

Plasmodium falciparum plasmepsin V (PfPMV) is an essential aspartic protease required for parasite survival, thus, considered as a potential drug target. This study reports the first detailed structural analysis and molecular dynamics simulation of PfPMV as an apoenzyme and its complexes with the substrate PEXEL as well as with the inhibitor saquinavir. The presence of pro-peptide in PfPMV may not structurally hinder the formation of a functionally competent catalytic active site.

The prosegment catalyzes native folding of Plasmodium falciparum plasmepsin II.

Plasmepsin II is a malarial pepsin-like aspartic protease produced as a zymogen containing an N-terminal prosegment domain that is removed during activation. Despite structural similarities between active plasmepsin II and pepsin, their prosegments adopt different conformations in the respective zymogens. In contrast to pepsinogen, the proplasmepsin II prosegment is 80 residues longer, contains a transmembrane region and is non-essential for recombinant expression in an active form, thus calling into question the prosegment's precise function.

A highly effective and versatile technology for the isolation of RNAs from grapevines and other woody perennials for use in virus diagnostics.

Background: Isolation of pure RNA from woody perennials, especially fruit crops such as grapevine rich in complex secondary metabolites, has remained very challenging. Lack of effective RNA isolation technology has resulted in difficulties in viral diagnosis and discovery as well as studies on many biological processes of these highly important woody plants. It is imperative to develop and refine methodologies with which large amounts of pure nucleic acids can be readily isolated from woody perennials.

Subcellular localization and membrane association of the replicase protein of grapevine rupestris stem pitting-associated virus, family Betaflexiviridae.

As a member of the newly established Betaflexiviridae family, grapevine rupestris stem pitting-associated virus (GRSPaV) has an RNA genome containing five ORFs. ORF1 encodes a putative replicase polyprotein typical of the alphavirus superfamily of positive-strand ssRNA viruses. Several viruses of this superfamily have been demonstrated to replicate in structures designated viral replication complexes associated with intracellular membranes.

The zymogen of plasmepsin V from Plasmodium falciparum is enzymatically active.

Plasmepsin V, a membrane-bound aspartic protease present in Plasmodium falciparum, is involved in the export of malaria parasite effector proteins into host erythrocytes and therefore is a potential target for antimalarial drug development. The present study reports the bacterial recombinant expression and initial characterization of zymogenic and mature plasmepsin V. A 484-residue truncated form of proplasmepsin (Glu37-Asn521) was fused to a fragment of thioredoxin and expressed as inclusion bodies.

Newly developed quantitative transactivation system shows difference in activation by Vitis CBF transcription factors on DRE/CRT elements.

Background: Agroinfiltration-based transactivation systems can determine if a protein functions as a transcription factor, and via which promoter element. However, this activation is not always a yes or no proposition. Normalization for variation in plasmid delivery into plant cells, sample collection and protein extraction is desired to allow for a quantitative comparison between transcription factors or promoter elements.

Structural insights into the activation and inhibition of histo-aspartic protease from Plasmodium falciparum.

Histo-aspartic protease (HAP) from Plasmodium falciparum is a promising target for the development of novel antimalarial drugs. The sequence of HAP is highly similar to those of pepsin-like aspartic proteases, but one of the two catalytic aspartates, Asp32, is replaced with histidine. Crystal structures of the truncated zymogen of HAP and of the complex of the mature enzyme with inhibitor KNI-10395 have been determined at 2.

The native conformation of plasmepsin II is kinetically trapped at neutral pH.

Plasmepsin II (PMII), an aspartic protease from the malarial parasite Plasmodium falciparum, represents a model for understanding protease structure/function relationships due to its unique structure and properties. The present study undertook a thermodynamic and kinetic analysis of the PMII folding mechanism and a pH stability profile. Differential scanning calorimetry revealed that the native state of PMII (Np) was irreversibly unfolded, and in the pH range of 6.

Crystal structures of the free and inhibited forms of plasmepsin I (PMI) from Plasmodium falciparum.

Plasmepsin I (PMI) is one of the four vacuolar pepsin-like proteases responsible for hemoglobin degradation by the malarial parasite Plasmodium falciparum, and the only one with no crystal structure reported to date. Due to substantial functional redundancy of these enzymes, lack of inhibition of even a single plasmepsin can defeat efforts in creating effective antiparasitic agents. We have now solved crystal structures of the recombinant PMI as apoenzyme and in complex with the potent peptidic inhibitor, KNI-10006, at the resolution of 2.

Characterization of the monomer-dimer equilibrium of recombinant histo-aspartic protease from Plasmodium falciparum.

Histo-aspartic protease (HAP) from Plasmodium falciparum is an intriguing aspartic protease due to its unique structure. Our previous study reported the first recombinant expression of soluble HAP, in its truncated form (lys77p-Leu328) (p denotes prosegment), as a thioredoxin (Trx) fusion protein Trx-tHAP. The present study found that the recombinant Trx-tHAP fusion protein aggregated during purification which could be prevented through the addition of 0.

Crystal structures of the histo-aspartic protease (HAP) from Plasmodium falciparum.

The structures of recombinant histo-aspartic protease (HAP) from malaria-causing parasite Plasmodium falciparum as apoenzyme and in complex with two inhibitors, pepstatin A and KNI-10006, were solved at 2.5-, 3.3-, and 3.

The catalytic significance of the proposed active site residues in Plasmodium falciparum histoaspartic protease.

Alanine mutations of the proposed catalytically essential residues in histoaspartic protease (HAP) (H34A, S37A and D214A) were generated to investigate whether: (a) HAP is a serine protease with a catalytic triad of His34, Ser37 and Asp214 [Andreeva N, Bogdanovich P, Kashparov I, Popov M & Stengach M (2004) Proteins55, 705-710]; or (b) HAP is a novel protease with Asp214 acting as both the acid and the base during substrate catalysis with His34 providing critical stabilization [Bjelic S & Aqvist J (2004) Biochemistry43, 14521-14528]. Our results indicated that recombinant wild-type HAP, S37A and H34A were capable of autoactivation, whereas D214A was not. The inability of D214A to autoactivate highlighted the importance of Asp214 for catalysis.

Expression and enzymatic characterization of the soluble recombinant plasmepsin I from Plasmodium falciparum.

The plasmepsins are involved in the degradation of host cell hemoglobin during malaria infection. Plasmepsin I (PM I) initiates the degradative process, and has been suggested as an attractive target for the treatment of malaria. The production of active recombinant PM I, however, has been challenging.

CBF4 is a unique member of the CBF transcription factor family of Vitis vinifera and Vitis riparia.

The CBF/DREB1 transcription factors control an important pathway for increased freezing and drought tolerance in plants. We report here the isolation of one CBF/DREB1-like gene, CBF4, from both freezing-tolerant wild grape (Vitis riparia) and freezing-sensitive cultivated grape (Vitis vinifera). The deduced protein in V.