Marc Fuchs

Transgenic plums (Prunus domestica L.) express the plum pox virus coat protein gene

SummaryPlum hypocotyl slices were transformed with the coat protein (CP) gene of plum pox virus (PPV-CP) following cocultivation with Agrobacterium tumefaciens containing the plasmid pGA482GG/PPVCP-33. This binary vector carries the PPV-CP gene construct, as well as the chimeric neomycin phosphotransferase and β-glucuronidase genes. Integration and expression of the transferred genes into regenerated plum plants was verified through kan resistance, GUS assays, and PCR amplification of the PPV-CP gene. Twenty-two transgenic clones were identified from approximately 1800 hypocotyl slices. DNA, mRNA, and protein analyses of five transgenic plants confirmed the integration of the engineered CP gene, the accumulation of CP mRNA and of PPV-CP-immunoreactive protein. CP mRNA levels ranged from high to undetectable levels, apparently correlated with gene structure, as indicated by DNA blot analysis. Western analysis showed that transgenic plants produced amounts of CP which generally correlated with amounts of detected mRNA.

Complete nucleotide sequence and genetic organization of grapevine fanleaf nepovirus RNA1

The nucleotide sequence of the genomic RNA1, 7342 nucleotides (nt) of grapevine fanleaf virus strain F13 (GFLV-F13) has been determined from cDNA clones. The complete sequence contained only one long open reading frame (ORF) of 6852 nucleotides extending from nucleotide 243 to 7101. The putative polyprotein encoded by this ORF is 2284 amino acids in length with an Mr of 253K. The location of genome-linked protein and comparison of the primary structure of the 253K polyprotein to that of other closely related viral proteins of the picronavirus-like family allows the proposal of a scheme for the genetic organization of GFLV-F13 RNA1. The primary structure of the polyprotein includes a putative RNA-dependent RNA polymerase of 92K and a cysteine protease of 25K. This protease shares not only major structural homologies, particularly in the substrate-binding pocket, with the trypsin-like serine proteases of other picorna-like viruses, but also their specificity in terms of cleavage. The large region of Mr 133K upstream of the VPg was found to contain at least two domains, one of which could be easily aligned with the NTP-binding sequence pattern and another which may have the characteristics of a protease cofactor. Thus, the 253K protein possesses the same general genetic organization as the corresponding protein of other picorna-like viruses.

Grapevine leafroll-associated virus 3

Grapevine leafroll disease (GLD) is one of the most important grapevine viral diseases affecting grapevines worldwide. The impact on vine health, crop yield, and quality is difficult to assess due to a high number of variables, but significant economic losses are consistently reported over the lifespan of a vineyard if intervention strategies are not implemented. Several viruses from the family Closteroviridae are associated with GLD. However, Grapevine leafroll-associated virus 3 (GLRaV-3), the type species for the genus Ampelovirus, is regarded as the most important causative agent. Here we provide a general overview on various aspects of GLRaV-3, with an emphasis on the latest advances in the characterization of the genome. The full genome of several isolates have recently been sequenced and annotated, revealing the existence of several genetic variants. The classification of these variants, based on their genome sequence, will be discussed and a guideline is presented to facilitate future comparative studies. The characterization of sgRNAs produced during the infection cycle of GLRaV-3 has given some insight into the replication strategy and the putative functionality of the ORFs. The latest nucleotide sequence based molecular diagnostic techniques were shown to be more sensitive than conventional serological assays and although ELISA is not as sensitive it remains valuable for high-throughput screening and complementary to molecular diagnostics. The application of next-generation sequencing is proving to be a valuable tool to study the complexity of viral infection as well as plant pathogen interaction. Next-generation sequencing data can provide information regarding disease complexes, variants of viral species, and abundance of particular viruses. This information can be used to develop more accurate diagnostic assays. Reliable virus screening in support of robust grapevine certification programs remains the cornerstone of GLD management.

Complete Genome Sequence of a New Circular DNA Virus from Grapevine

ABSTRACT A novel circular DNA virus sequence is reported from grapevine. The corresponding genomic organization, coding potential, and conserved origin of replication are similar to those of members of the family Geminiviridae, but the genome of 3,206 nucleotides is 4% larger than the largest reported geminiviral genome and shares only 50% overall sequence identity.

Resistance to squash mosaic comovirus in transgenic squash plants expressing its coat protein genes

The approach of pathogen-derived resistance was investigated as a means to develop squash mosaic comovirus (SqMV)-resistant cucurbits. Transgenic squash lines with both coat protein (CP) genes of the melon strain of SqMV were produced and crossed with nontransgenic squash. Further greenhouse, screenhouse and field tests were done with R1 plants from three independent lines that showed susceptible, recovery, or resistant phenotypes after inoculations with SqMV. Nearly all inoculated plants of the resistant line (SqMV-127) were resistant under greenhouse and field conditions and less so under screenhouse conditions. Plants of the recovery phenotype line (SqMV-3) were susceptible when inoculated at the cotyledon stage but leaves that developed later did not show symptoms. The susceptible line (SqMV-22) developed symptoms that persisted and spread throughout the plant. Plants were also analyzed for transcription rates of the CP transgenes and steady state transgene RNA levels. Results showed that the resistant line SqMV-127 displayed post-transcriptional silencing of the CP transgene as evidenced by high transcription rates but concomitant low accumulation of transgene transcripts. This is the first report on the development of transgenic squash that are resistant to SqMV.

Comparative virus resistance and fruit yield of transgenic squash with single and multiple coat protein genes

Five transgenic squash lines expressing coat protein (CP) genes from cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic virus 2 potyvirus (WMV 2) were analyzed in the field for their reaction to mixed infections by these three viruses and for fruit production. Test plants were exposed to natural inoculations via aphids in trials simulating the introduction of viruses by secondary spread from mechanically infected susceptible border row plants. Plants of transgenic line CZW-3 expressing the CP genes from CMV, ZYMV, and WMV 2 displayed the highest level of resistance with no systemic infection, although 64% exhibited localized chlorotic dots which were mainly confined to older leaves. CZW-3 plants had a 50-fold increase in marketable yield compared to controls and the highest predicted cash returns. Plants of transgenic line ZW-20 expressing the CP genes from ZYMV and WMV 2 displayed high levels of resistance to these two potyviruses, but 22% became infected by CMV. However, ZW-20 plants provided a 40-fold increase in marketable yield relative to controls and good estimated cash returns. Three transgenic lines expressing single CP genes from either ZYMV (line Z-33), WMV 2 (line W-164) or CMV (line C-14) developed systemic symptoms similar to those of controls but showed a delay of 2 to 4 weeks before the onset of disease. Plants of transgenic line Z-33 were highly resistant to ZYMV but not to WMV 2 and CMV. Interestingly, Z-33 plants had a 20-fold increase in marketable yield compared to controls and some predicted cash returns if market sale prices were high. This study indicates that virus-resistant transgenic lines are economically viable even if they are affected by viruses other than those to which they are resistant.

Cantaloupe line CZW-30 containing coat protein genes of cucumber mosaic virus, zucchini yellow mosaic virus, and watermelon mosaic virus-2 is resistant to these three viruses in the field

Cantaloupe line CZW-30 containing coat protein gene constructs of cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic virus 2 potyvirus (WMV-2) was investigated in the field over two consecutive years for resistance to infections by CMV, ZYMV, and/or WMV-2. Resistance was evaluated under high disease pressure achieved by mechanical inoculations and/or natural challenge inoculations by indigenous aphid vectors. Across five different trials, homozygous plants were highly resistant in that they never developed systemic symptoms as did the nontransformed plants but showed few symptomatic leaves confined close to the vine tips. Hemizygous plants exhibited a significant delay (2–3 weeks) in the onset of disease compared to control plants but had systemic symptoms 9–10 weeks after transplanting to the field. Importantly, ELISA data revealed that transgenic plants reduced the incidence of mixed infections. Only 8% of the homozygous and 33% of the hemizygous plants were infected by two or three viruses while 99% of the nontransformed plants were mixed infected. This performance is of epidemiological significance. In addition, control plants were severely stunted (44% reduction in shoot length) and had poor fruit yield (62% loss) compared to transgenic plants, and most of their fruits (60%) were unmarketable. Remarkably, hemizygous plants yielded 7.4 times more marketable fruits than control plants, thus suggesting a potential commercial performance. This is the first report on extensive field trials designed to assess the resistance to mixed infection by CMV, ZYMV, and WMV-2, and to evaluate the yield of commercial quality cantaloupes that are genetically engineered.

Economic Impact of Grapevine Leafroll Disease on Vitis vinifera cv. Cabernet franc in Finger Lakes Vineyards of New York

Leafroll disease is one of the most important virus diseases of grapevines worldwide. It reduces yields, delays fruit ripening, reduces soluble solids, and increases titratable acidity in fruit juice. This study uses a net present value (NPV) approach over a 25-year lifespan of a vineyard to examine the economic impact of grapevine leafroll disease (GLRD) on Vitis vinifera cv. Cabernet franc in Finger Lakes vineyards of New York. It identifies optimal disease control options under several scenarios of disease prevalence, yield reduction, and fruit quality effects. The estimated economic impact of GLRD ranges from approximately

Onion Thrips (Thysanoptera: Thripidae): A Global Pest of Increasing Concern in Onion

25,000 (for a 30% yield reduction and no grape quality penalty) to

Capulavirus and Grablovirus: two new genera in the family Geminiviridae

40,000 (for a 50% yield reduction and a 10% penalty for poor fruit quality) per hectare in the absence of any control measure. The per hectare impact of GLRD can be substantially reduced to