Richard W. Jones

Alternate intron processing of family 5 endoglucanase transcripts from the genus Phytophthora.

Twenty-one homologs of family 5 endo-(1–4)-β-glucanase genes (EGLs) were identified and characterized in the oomycete plant pathogens Phytophthora infestans, P. sojae, and P. ramorum, providing the first comprehensive analysis of this family in Phytophthora. Phylogenetic analysis revealed that these genes constitute a unique eukaryotic group, with closest similarity to bacterial endoglucanases. Many of the identified EGL copies were clustered in a few genomic regions, and contained from zero to three introns. Using reverse transcription PCR to study in vitro and in planta gene expression levels of P. sojae, we detected partially processed RNA transcripts retaining one or more of their introns. In some cases, the positions of intron/exon splicing sites were also found to be variable. The relative proportions of these transcripts remain apparently unchanged under various growing conditions, but differ among orthogolous copies of the three Phytophthora species. The alternate processing of introns in this group of EGLs generates both coding and non-coding RNA isoforms. This is the first report on Phytophthora family 5 endoglucanases, and the first record for alternative intron processing of oomycete transcripts.

Characterization of the glucose-6-phosphate isomerase gene in Phytophthora infestans reveals the presence of multiple alleles

Glucose-6-phosphate isomerase (GPI) plays a key role in both glycolysis and gluconeogenesis. Isoforms of GPI are common, and therefore, its isozyme pattern is widely used to characterize isolates of Phytophthora infestans. Despite the importance of GPI in P. infestans studies, the gene encoding this enzyme has not yet been characterized. Furthermore, it has been suggested that P. infestans contains multiple copies of the gene but this hypothesis remains to be demonstrated. We have cloned and characterized GPI in various isolates of P. infestans as well as in several species of the genus Phytophthora. The gene contains 1671bp and encodes a protein with a predicted molecular weight of 60.8kDa. Multiple different alleles were identified and Southern analysis indicated certain P. infestans isolates carry several copies of the gene. Phylogenetic analysis revealed that P. infestans GPI is most closely related to sequences from Toxoplasma gondii, Arabidopsis thaliana, and Clarkia lewisii.

Gene silencing indicates a role for potato endoglucanase inhibitor protein in germplasm resistance to late blight

Durable resistance to late blight is believed to be dependent on genes other than R genes. Reduced rates of lesion expansion, as found in the germplasm B 0767-2, can provide durable resistance by limiting tissue damage and reducing pathogen populations. We have tested the role of the proteinaceous xyloglucan-specific endoglucanase inhibitor (XEIP) in limiting late blight lesion expansion in B 0767-2. Plant material was silenced for the XEIP using localized infiltration ofAgrobacterium tumefaciens harboring pCAMBIA 1302 containing an antisense copy of XEIP. Plant cell transformations with this construct induced effective silencing as indicated by a reduction of up to 80% in XEIP transcript levels. Silencing of XEIP resulted in a marked increase in lesion size and water soaking, indicating that the inhibitor protein plays a role in limiting lesion expansion in B 0767-2.ResumenSe cree que la resistencia durable al tizón tardío es dependiente de genes diferentes a los genes R. Tasas reducidas de expansión de las lesiones como las encontradas en el germoplasma B 0767-2 pueden proporcionar resistencia durable, limitando el daño al tejido y reduciendo la población del patógeno. Hemos evaluado el rol del inhibidor endogluconasa xyloglucan proteinaceo específico (XEIP) para limitar la expansión de la lesión de tizón tardío en B 0767-2. El material vegetal quedó silenciado para el XEIP por infiltración localizada deAgrobacterium tumefaciens produciendo pCAMBIA 1302 que contiene una copia de XEIP en sentido contrario. Las transformaciones celulares de la planta con este “construct” indujeron un silenciado efectivo, indicado por una reducción de hasta en un 80% en los niveles de trascripción del XEIP. El silenciamiento del XEIP dio como resultado un marcado crecimiento del tamaño de la lesión y la absorción de agua, indicando que la proteína del inhibidor limita la expansión de la lesión en B 0767-2.

Prosystemin-antimicrobial-peptide fusion reduces tomato late blight lesion expansion

Antimicrobial peptides offer a new method for controlling pathogens, however, many promising peptides are too small for direct production in plants. A protein delivery system was developed based on a proteolytic mechanism used by Solanaceous plants to produce the very small (18 amino acid) signaling peptide systemin from the polypeptide prosystemin. Fusion of the gene encoding the 23 kDa protein prosystemin with the antimicrobial peptide (pep11) sequence, replacing the systemin sequence, allowed for expression in transgenic tomato plants. Six days after inoculation with the late blight pathogen Phytophthora infestans, detached leaflets of transgenic tomato (Rutgers) exhibited a reduction in lesion size of at least 50 percent.

Properties of the Macrophomina phaseolina Endoglucanase (EGL 1) Gene Product in Bacterial and Yeast Expression Systems

Functional expression of a β-d-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413. Optimal activity of the E. coli-expressed enzyme was found at incubation temperatures of 60°C, whereas the enzyme activity was optimal at 40°C when expressed by S. cerevisiae. Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0. Yeast expression resulted in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa. The hyperglycosylated protein had reduced enzyme activity, indicating that E. coli is a preferred vehicle for production scale-up.

Multiple Copies of Genes Encoding XEGIPs are Harbored in an 85-kB Region of the Potato Genome

Proteinaceous inhibitors of plant cell wall degrading enzymes can provide a first level of defense from invasive phytopathogens. One recently discovered inhibitor is the xyloglucans-specific endoglucanase inhibitor protein (XEGIP) originally found in tomato. This inhibitor protein has since been found in many solanaceous plants as well as other dicots. As xyloglucans play a major role in maintaining cell wall structure in dicots, protection of xyloglucan is critical. Thus far, only a single potato XEGIP had been identified (AY321357), even though a major pathogen of potato, Phytophthora infestans, has multiple copies of xyloglucan-specific endoglucanase. We now report that potato has nine additional copies of XEGIP, clustered together on a small region of chromosome one. They are located adjacent to the previously described potato XEGIP and represent one of the highest degrees of gene duplication relative to any other potato inhibitor. Synteny with tomato indicates this duplication may have occurred before speciation of Solanum.

Herbivory responsive C2H2 zinc finger transcription factor protein StZFP2 from potato.

While C2H2 zinc finger transcription factors (TF) are often regulated by abiotic stress, their role during insect infestation has been overlooked. This study demonstrates that the transcripts of the zinc finger transcription factors StZFP1 and StZFP2 are induced in potato (Solanum tuberosum L.) upon infestation by either the generalist tobacco hornworm (THW, Manduca sexta L.) or the specialist Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). StZFP1 has been previously characterized as conferring salt tolerance to transgenic tobacco and its transcript is induced by Phytophthora infestans and several abiotic stresses. StZFP2 has not been characterized previously, but contains the hallmarks of a C2H2 zinc finger TF, with two conserved zinc finger domains and DLN motif, which encodes a transcriptional repressor domain. Expression studies demonstrate that StZFP2 transcript is also induced by tobacco hornworm and Colorado potato beetle. These observations expand the role of the C2H2 transcription factor in potato to include the response to chewing insect pests.

A cellulose binding domain protein restores female fertility when expressed in transgenic Bintje potato

BackgroundExpression of a gene encoding the family 1 cellulose binding domain protein CBD1, identified in the cellulosic cell wall of the potato late blight pathogen Phytophthora infestans, was tested in transgenic potato to determine if it had an influence on plant cell walls and resistance to late blight.ResultsMultiple regenerants of potato (cv. Bintje) were developed and selected for high expression of CBD 1 transcripts. Tests with detached leaflets showed no evidence of increased or decreased resistance to P. infestans, in comparison with the blight susceptible Bintje controls, however, changes in plant morphology were evident in CBD 1 transgenics. Plant height increases were evident, and most importantly, the ability to produce seed berries from a previously sterile cultivar. Immunolocalization of CBD 1 in seed berries revealed the presence throughout the tissue. While Bintje control plants are male and female sterile, CBD 1 transgenics were female fertile. Crosses made using pollen from the late blight resistant Sarpo Mira and transgenic CBD1 Bintje as the female parent demonstrated the ability to introgress P. infestans targeted resistance genes, as well as genes responsible for color and tuber shape, into Bintje germplasm.ConclusionsA family 1 cellulose-binding domain (CBD 1) encoding gene from the potato late blight pathogen P. infestans was used to develop transgenic Bintje potato plants. Transgenic plants became female fertile, allowing for a previously sterile cultivar to be used in breeding improvement. Selection for the absence of the CBD transgene in progeny should allow for immediate use of a genetically enhanced material. Potential for use in other Solanaceous crops is proposed.

A Small Cellulose-Binding-Domain Protein (CBD1) in Phytophthora is Highly Variable in the Non-binding Amino Terminus

The small cellulose-binding-domain protein CBD1 is tightly bound to the cellulosic cell wall of the plant pathogenic stramenopile Phytophthora infestans. Transgene expression of the protein in potato plants also demonstrated binding to plant cell walls. A study was undertaken using 47 isolates of P. infestans from a worldwide collection, along with 17 other Phytophthora species and a related pathogen Plasmopara halstedii, to determine if the critical cell wall protein is subject to amino acid variability. Within the amino acid sequence of the secreted portion of CBD 1, encoded by the P. infestans isolates, 30 were identical with each other, and with P. mirabilis. Four isolates had one amino acid difference, each in a different location, while one isolate had two amino acid substitutions. The remaining 13 isolates had five amino acid changes that were each in identical locations (D17/G, D31/G, I32/S, T43/A, and G50/A), suggesting a single origin. Comparison of P. infestans CBD1 with other Phytophthora species identified extensive amino acid variation among the 60 amino acids at the amino terminus of the protein, and a high level of conservation from G61, where the critical cellulose-binding domain sequences begin, to the end of the protein (L110). While the region needed to bind to cellulose is conserved, the region that is available to interact with other cell wall components is subject to considerable variation, a feature that is evident even in the related genus Plasmopara. Specific changes can be used in determining intra- and inter-species relatedness. Application of this information allowed for the design of species-specific primers for PCR detection of P. infestans and P. sojae, by combining primers from the highly conserved and variable regions of the CBD1 gene.

Application of succulent plant leaves for Agrobacterium infiltration-mediated protein production

When expressing plant cell wall degrading enzymes in the widely used tobacco (Nicotiana benthamiana) after Agrobacterium infiltration, difficulties arise due to the thin leaf structure. Thick leaved succulents, Kalanchoe blossfeldiana and Hylotelephium telephium, were tested as alternatives. A xyloglucanase, as well as a xyloglucanase inhibitor protein was successfully produced.