Field test of Easter lilies transformed with a rice cystatin gene for root lesion nematode resistance.

Easter lilies, Lilium longiflorum cv. Nellie White are a staple of the floral industry. In the U.S. most of the Easter lilies are grown in Oregon and California along the coast where there is a micro climate that is favorable to growth of lilies. The main pest when growing lilies in the field is Pratylenchus penetrans, the root lesion nematode. Easter lilies are one of the most expensive crops to produce because of the cost of chemicals used to control P. penetrans and other pathogens that infect the lilies. Our previous study had shown that transgenic Easter lilies containing a rice cystatin gene (Oc-IΔD86 that has a deleted Asp86) were resistant to P. penetrans in vitro. This study examined growth characteristics of five independently transformed lines of the cystatin Easter lilies compared to non-transformed Nellie White for three seasons in the field in Brookings, Oregon. Liles grown in three soil chemical treatments 1) preplant fumigation, 2) preplant fumigation plus at plant organophosphate, and 3) at plant organophosphate were compared to those grown in nontreated soil. Growth characteristics evaluated included: time of shoot emergence, survival of plants, size of plants, visual ratings of plant health, basal roots and stem roots, weight of foliage and roots, and number and size of bulblets that developed on stems. Nematodes were counted following their extraction from the roots. While not totally resistant, when planted in the field, transformed lines demonstrated and maintained a degree of resistance to lesion nematode over two growing seasons and displayed desirable growth and quality characteristics similar to non-transformed lilies.

Identification and characterization of the first pectin methylesterase gene discovered in the root lesion nematode Pratylenchus penetrans.

Similar to other plant-parasitic nematodes, root lesion nematodes possess an array of enzymes that are involved in the degradation of the plant cell wall. Here we report the identification of a gene encoding a cell wall-degrading enzyme, pectin methylesterase PME (EC 3.1.1.11), in the root lesion nematode Pratylenchus penetrans. Both genomic and coding sequences of the gene were cloned for this species, that included the presence of four introns which eliminated a possible contamination from bacteria. Expression of the Pp-pme gene was localized in the esophageal glands of P. penetrans as determined by in situ hybridization. Temporal expression of Pp-pme in planta was validated at early time points of infection. The possible function and activity of the gene were assessed by transient expression of Pp-pme in plants of Nicotiana benthamiana plants via a Potato virus X-based vector. To our knowledge, this is the first report on identification and characterization of a PME gene within the phylum Nematoda.

Identification of candidate effector genes of Pratylenchus penetrans.

Pratylenchus penetrans is one of the most important species of root lesion nematodes (RLNs) because of its detrimental and economic impact in a wide range of crops. Similar to other plant-parasitic nematodes (PPNs), P. penetrans harbours a significant number of secreted proteins that play key roles during parasitism. Here, we combined spatially and temporally resolved next-generation sequencing datasets of P. penetrans to select a list of candidate genes aimed at the identification of a panel of effector genes for this species. We determined the spatial expression of transcripts of 22 candidate effectors within the oesophageal glands of P. penetrans by in situ hybridization. These comprised homologues of known effectors of other PPNs with diverse putative functions, as well as novel pioneer effectors specific to RLNs. It is noteworthy that five of the pioneer effectors encode extremely proline-rich proteins. We then combined in situ localization of effectors with available genomic data to identify a non-coding motif enriched in promoter regions of a subset of P. penetrans effectors, and thus a putative hallmark of spatial expression. Expression profiling analyses of a subset of candidate effectors confirmed their expression during plant infection. Our current results provide the most comprehensive panel of effectors found for RLNs. Considering the damage caused by P. penetrans, this information provides valuable data to elucidate the mode of parasitism of this nematode and offers useful suggestions regarding the potential use of P. penetrans-specific target effector genes to control this important pathogen.

Characterization of Lilium longiflorum cv. 'Nellie White' Infection with Root-lesion Nematode Pratylenchus penetrans by Bright-field and Transmission Electron Microscopy.

Lilium longiflorum cv. Nellie White, commonly known as Easter lily, is an important floral crop with an annual wholesale value of over $26 million in the United States. The root-lesion nematode, Pratylenchus penetrans, is a major pest of lily due to the significant root damage it causes. In this study, we investigated the cytological aspects of this plant-nematode interaction using bright-field and transmission electron microscopy. We took advantage of an in vitro culture method to multiply lilies and follow the nematode infection over time. Phenotypic reactions of roots inoculated with P. penetrans were evaluated from 0 to 60 d after nematode infection. Symptom development progressed from initial randomly distributed discrete necrotic areas to advanced necrosis along entire roots of each inoculated plant. A major feature characterizing this susceptible host response to nematode infection was the formation of necrosis, browning, and tissue death involving both root epidermis and cortical cells. Degradation of consecutive cell walls resulted in loss of cell pressure, lack of cytoplasmic integrity, followed by cell death along the intracellular path of the nematode's migration. Pratylenchus penetrans was never seen in the vascular cylinder as the layer of collapsed endodermal cells presumably blocked the progression of nematodes into this area of the roots. This study presents the first detailed cytological characterization of P. penetrans infection of Easter lily plants.

Symptom Development in Response to Combined Infection of In Vitro-grown Lilium longiflorum with Pratylenchus penetrans and Soilborne Fungi Collected from Diseased Roots of Field-grown Lilies.

Eight fungal isolates (ELRF 1 to 8) were recovered from necrotic roots of Easter lilies, Lilium longiflorum cv. Nellie White, grown in a field in the U.S. Pacific Northwest. The eight fungal isolates were identified by sequencing and molecular phylogenetic analyses based on their ITS rDNA region. Five isolates were identified as Fusarium oxysporum, two as F. tricinctum, and one as Rhizoctonia sp. AG-I. This constitutes the first report of Rhizoctonia sp. AG-I infecting lilies worldwide and the first report of F. tricinctum infecting lilies in the United States. To study and validate their pathogenicity, pure cultures of each isolate were used to infect the roots of Easter lily plants growing in vitro. In addition, Easter lily plants growing in vitro were infected either with or without Pratylenchus penetrans, the root lesion nematode, prior to placing a culture plug of fungus 1 cm from a lily root. Pratylenchus penetrans is a nematode species commonly found in the sampled fields. The presence of both nematode and Rhizoctonia sp. AG-I isolate ELRF 3 in infected lilies was evaluated by molecular analyses, confirming the infection of roots 3 days after inoculation, prior to development of disease symptoms. Necrosis and root rot developed more rapidly with all eight fungal isolates when there had been prior infection with P. penetrans, the major nematode parasitizing Easter lily roots in the field in Oregon.

The Pratylenchus penetrans Transcriptome as a Source for the Development of Alternative Control Strategies: Mining for Putative Genes Involved in Parasitism and Evaluation of in planta RNAi.

The root lesion nematode Pratylenchus penetrans is considered one of the most economically important species within the genus. Host range studies have shown that nearly 400 plant species can be parasitized by this species. To obtain insight into the transcriptome of this migratory plant-parasitic nematode, we used Illumina mRNA sequencing analysis of a mixed population, as well as nematode reads detected in infected soybean roots 3 and 7 days after nematode infection. Over 140 million paired end reads were obtained for this species, and de novo assembly resulted in a total of 23,715 transcripts. Homology searches showed significant hit matches to 58% of the total number of transcripts using different protein and EST databases. In general, the transcriptome of P. penetrans follows common features reported for other root lesion nematode species. We also explored the efficacy of RNAi, delivered from the host, as a strategy to control P. penetrans, by targeted knock-down of selected nematode genes. Different comparisons were performed to identify putative nematode genes with a role in parasitism, resulting in the identification of transcripts with similarities to other nematode parasitism genes. Focusing on the predicted nematode secreted proteins found in this transcriptome, we observed specific members to be up-regulated at the early time points of infection. In the present study, we observed an enrichment of predicted secreted proteins along the early time points of parasitism by this species, with a significant number being pioneer candidate genes. A representative set of genes examined using RT-PCR confirms their expression during the host infection. The expression patterns of the different candidate genes raise the possibility that they might be involved in critical steps of P. penetrans parasitism. This analysis sheds light on the transcriptional changes that accompany plant infection by P. penetrans, and will aid in identifying potential gene targets for selection and use to design effective control strategies against root lesion nematodes.

Expression of a cystatin transgene can confer resistance to root lesion nematodes in Lilium longiflorum cv. 'Nellie White'.

Lilium longiflorum cv. 'Nellie White' assumes a great economic importance as cut flowers, being one of the most valuable species (annual pot plants value above $20,000,000) in terms of wholesales in the US. The root lesion nematode Pratylenchus penetrans (RLN) constitutes one of the main pests for lily producers due to the significant root damage it causes. Our efforts have focused on the generation of soybean hairy roots (as a transient test model) and stable transgenic lilies overexpressing a modified rice cystatin (Oc-IΔD86) transgene and challenged with root lesion nematodes. Lily transformation was achieved by gene gun co-bombardment using both a pBluescript-based vector containing the cystatin gene and pDM307 that contains a bar gene for phosphinothricin selection. Both soybean hairy roots and lilies overexpressing the OcIΔD86 transgene exhibited enhanced resistance to RLN infection by means of nematode reduction up to 75 ± 5% on the total number of nematodes. In addition, lily plants overexpressing OcIΔD86 displayed an increase of plant mass and better growth performance in comparison to wild-type plants, thereby demonstrating an alternative strategy for increasing the yield and reducing nematode damage to this important floral crop.

The 5'UTR-intron of the Gladiolus polyubiquitin promoter GUBQ1 enhances translation efficiency in Gladiolus and Arabidopsis.

BACKGROUND: There are many non-cereal monocots of agronomic, horticultural, and biofuel importance. Successful transformation of these species requires an understanding of factors controlling expression of their genes. Introns have been known to affect both the level and tissue-specific expression of genes in dicots and cereal monocots, but there have been no studies on an intron isolated from a non-cereal monocot. This study characterizes the levels of GUS expression and levels of uidA mRNA that code for ß-glucuronidase (GUS) expression in leaves of Gladiolus and Arabidopsis using GUBQ1, a polyubiquitin promoter with a 1.234 kb intron, isolated from the non-cereal monocot Gladiolus, and an intronless version of this promoter. RESULTS: Gladiolus and Arabidopsis were verified by Southern hybridization to be transformed with the uidA gene that was under control of either the GUBQ1 promoter (1.9 kb), a 5' GUBQ1 promoter missing its 1.234 kb intron (0.68 kb), or the CaMV 35 S promoter. Histochemical staining showed that GUS was expressed throughout leaves and roots of Gladiolus and Arabidopsis with the 1.9 kb GUBQ1 promoter. GUS expression was significantly decreased in Gladiolus and abolished in Arabidopsis when the 5'UTR-intron was absent. In Arabidopsis and Gladiolus, the presence of uidA mRNA was independent of the presence of the 5'UTR-intron. The 5'-UTR intron enhanced translation efficiency for both Gladiolus and Arabidopsis. CONCLUSIONS: The GUBQ1 promoter directs high levels of GUS expression in young leaves of both Gladiolus and Arabidopsis. The 5'UTR-intron from GUBQ1 resulted in a similar pattern of ß-glucuronidase translation efficiency for both species even though the intron resulted in different patterns of uidA mRNA accumulation for each species.

Resistance to Cucumber mosaic virus in Gladiolus plants transformed with either a defective replicase or coat protein subgroup II gene from Cucumber mosaic virus.

Transgenic Gladiolus plants that contain either Cucumber mosaic virus (CMV) subgroup I coat protein, CMV subgroup II coat protein, CMV replicase, a combination of the CMV subgroups I and II coat proteins, or a combination of the CMV subgroup II coat protein and replicase genes were developed. These plants were multiplied in vitro and challenged with purified CMV isolated from Gladiolus using a hand-held gene gun. Three out of 19 independently transformed plants expressing the replicase gene under control of the duplicated CaMV 35S promoter were found to be resistant to CMV subgroup I. Three out of 21 independently transformed plants with the CMV subgroup II coat protein gene under control of the Arabidopsis UBQ3 promoter were resistant to CMV subgroup II. Eighteen independently transformed plants with either the CMV subgroup I coat protein or a combination of CMV subgroups I and II coat proteins were challenged and found to be susceptible to both CMV subgroups I or II. Virus resistant plants with the CMV replicase transgene expressed much lower RNA levels than resistant plants expressing the CMV subgroup II coat protein. This work will facilitate the evaluation of virus resistance in transgenic Gladiolus plants to yield improved floral quality and productivity.

Expression of a polyubiquitin promoter isolated from Gladiolus.

A polyubiquitin promoter (GUBQ1) including its 5'UTR and intron was isolated from the floral monocot Gladiolus because high levels of expression could not be obtained using publicly available promoters isolated from either cereals or dicots. Sequencing of the promoter revealed highly conserved 5' and 3' intron splicing sites for the 1.234 kb intron. The coding sequence of the first two ubiquitin genes showed the highest homology (87 and 86%, respectively) to the ubiquitin genes of Nicotiana tabacum and Oryza sativa RUBQ2. Transient expression following gene gun bombardment showed that relative levels of GUS activity with the GUBQ1 promoter were comparable to the CaMV 35S promoter in gladiolus, tobacco, rose, rice, and the floral monocot freesia. The highest levels of GUS expression with GUBQ1 were attained with Gladiolus. The full-length GUBQ1 promoter including 5'UTR and intron were necessary for maximum GUS expression in Gladiolus. The relative GUS activity for the promoter only was 9%, and the activity for the promoter with 5'UTR and 399 bp of the full-length 1.234 kb intron was 41%. Arabidopsis plants transformed with uidA under GUBQ1 showed moderate GUS expression throughout young leaves and in the vasculature of older leaves. The highest levels of transient GUS expression in Gladiolus have been achieved using the GUBQ1 promoter. This promoter should be useful for genetic engineering of disease resistance in Gladiolus, rose, and freesia, where high levels of gene expression are important.

Biolistic inoculation of gladiolus with cucumber mosaic cucumovirus.

A new method of inoculation of gladiolus with cucumber mosaic virus (CMV) was developed using the Bio-Rad Helios Gene Gun System. This method circumvents the traditional use of aphids to transmit CMV, a virus that is mechanically transmissible to many plant species but only with difficulty to gladiolus. Cartridges containing virus-coated gold microcarriers were prepared and the virus shot into Nicotianabenthamiana leaves and gladiolus corms and cormels. The biolistic procedure successfully transmitted three CMV isolates, two from serogroup I and one from serogroup II. Survival rates of two cultivars of gladiolus cormels and corms in sterile and non-sterile environments were compared. Infection rates of 100% were obtained when as little as 2 microg of virus was used in cartridge preparation. CMV remained viable after the cartridges were stored for many months at 4 degrees C.

Transgenic Gladiolus plants transformed with the bean yellow mosaic virus coat-protein gene in either sense or antisense orientation.

Transgenic Gladiolus plants transformed with the bean yellow mosaic virus (BYMV) coat-protein (CP) gene in either sense or antisense (AS) orientation were developed using biolistics. Four of the plants were confirmed to carry the CP gene in the sense orientation of the gene and seven plants in the AS orientation. Two of the CP plant lines and all of the AS lines showed DNA rearrangements of the transgene in addition to an intact copy of the transgene. The copy number ranged from one to nine. Of the 11 lines, eight had only one to four copies of the transgene. Transcription of the transgene occurred for three of the CP lines and five of the AS lines as determined by Northern hybridization. All 11 plant lines were challenged with BYMV using controlled aphid transmission. One month following aphid transmission, the transgenic plants were examined by immunoelectron microscopy for presence of the virus. Several transgenic plant lines containing either antiviral transgene showed a lower incidence of infection (percentage of plants infected as detected by immunoelectron microscopy) than the non-transformed plants. Most of the CP- and AS-transgenic plants that did not contain BYMV 1 month after challenge were found to contain BYMV the next season. It appeared that BYMV infection was delayed in the CP- and AS-transgenic lines but that the transgenes did not prevent eventual infection of BYMV. This is the first report of developing a floral bulb crop with antiviral genes to BYMV.