SGT1 Genes Research Articles

GGE Biplot-Based Transcriptional Analysis of 7 Genes Involved in Steroidal Glycoalkaloid Biosynthesis in Potato (Solanum tuberosum L.)

Steroidal glycoalkaloids (SGAs) are secondary metabolites that are closely associated with the sensory and processing qualities of potato tubers. GGE biplots are a widely used tool for analyzing crop breeding analysis. This study aimed to investigate the effect of light on SGA biosynthesis by employing GGE biplots to analyze the transcriptional gene expression of seven genes involved in the SGA biosynthesis pathway. Tubers of five different potato genotypes were incubated for 6, 12, and 24 h under red light. The expression levels of the seven genes were measured using qRT-PCR for analysis. Further analysis of the data was performed using GGE biplots. Our results indicated significantly higher expression levels for Pvs1, Sgt1, and Sgt3 genes than those of the remaining tested genes. Across the three red light illumination durations, Sgt3 showed high and stable expression, although it showed less stability across the different genotypes. Interestingly, the expression patterns of the seven genes were extremely similar for the 12 h and 24 h treatments. It was found that at least 6 h of red light illumination was required for optimal gene expression in all five genotypes, particularly in the genotype Zhuangshu-3 (DXY) after 24 h of treatment. Additionally, significant expression of the seven genes was observed in the L-6 genotype after 12 and 6 h of red light illumination. These results highlight that GGE biplots are an appropriate tool for analyzing and illustrating the differential expression profiles of the seven key genes involved in SGA biosynthesis in potato tubers. This study provides valuable insights into the biosynthesis and metabolism of SGAs in potatoes. Moreover, it demonstrates the potential application of GGE biplots in crop breeding and other research fields.

Genome-Wide Identification and Characterization of HSP90-RAR1-SGT1-Complex Members From Arachis Genomes and Their Responses to Biotic and Abiotic Stresses

The molecular chaperone complex HSP90-RAR1-SGT1 (HRS) plays important roles in both biotic and abiotic stress responses in plants. A previous study showed that wild peanut Arachis diogoi SGT1 (AdSGT1) could enhance disease resistance in transgenic tobacco and peanut. However, no systematic analysis of the HRS complex in Arachis has been conducted to date. In this study, a comprehensive analysis of the HRS complex were performed in Arachis. Nineteen HSP90, two RAR1 and six SGT1 genes were identified from the allotetraploid peanut Arachis hypogaea, a number close to the sum of those from the two wild diploid peanut species Arachis duranensis and Arachis ipaensis. According to phylogenetic and chromosomal location analyses, thirteen orthologous gene pairs from Arachis were identified, all of which except AhHSP90-A8, AhHSP90-B9, AdHSP90-9, and AiHSP90-9 were localized on the syntenic locus, and they shared similar exon-intron structures, conserved motifs and expression patterns. Phylogenetic analysis showed that HSP90 and RAR1 from dicot and monocot plants diverged into different clusters throughout their evolution. Chromosomal location analysis indicated that AdSGT1 (the orthologous gene of AhSGT1-B3 in this study) might provide resistance to leaf late spot disease dependent on the orthologous genes of AhHSP90-B10 and AhRAR1-B in the wild peanut A. diogoi. Several HRS genes exhibited tissue-specific expression patterns, which may reflect the sites where they perform functions. By exploring published RNA-seq data, we found that several HSP90 genes play major roles in both biotic and abiotic stress responses, especially salt and drought responses. Autoactivation assays showed that AhSGT1-B1 could not be used as bait for yeast two-hybrid (Y2H) library screening. AhRAR1 and AhSGT1 could strongly interact with each other and interact with AhHSP90-B8. The present study represents the first systematic analysis of HRS complex genes in Arachis and provides valuable information for functional analyses of HRS complex genes. This study also offers potential stress-resistant genes for peanut improvement.

Comparative transcriptome analysis of R3a and Avr3a-mediated defense responses in transgenic tomato.

Late blight caused by Phytophthora infestans is one of the most devastating diseases in potatoes and tomatoes. At present, several late blight resistance genes have been mapped and cloned. To better understand the transcriptome changes during the incompatible interaction process between R3a and Avr3a, in this study, after spraying DEX, the leaves of MM-R3a-Avr3a and MM-Avr3a transgenic plants at different time points were used for comparative transcriptome analysis. A total of 7,324 repeated DEGs were detected in MM-R3a-Avr3a plants at 2-h and 6-h, and 729 genes were differentially expressed at 6-h compared with 2-h. Only 1,319 repeated DEGs were found in MM-Avr3a at 2-h and 6-h, of which 330 genes have the same expression pattern. Based on GO, KEGG and WCGNA analysis of DEGs, the phenylpropanoid biosynthesis, plant-pathogen interaction, and plant hormone signal transduction pathways were significantly up-regulated. Parts of the down-regulated DEGs were enriched in carbon metabolism and the photosynthesis process. Among these DEGs, most of the transcription factors, such as WRKY, MYB, and NAC, related to disease resistance or endogenous hormones SA and ET pathways, as well as PR, CML, SGT1 gene were also significantly induced. Our results provide transcriptome-wide insights into R3a and Avr3a-mediated incompatibility interaction.

Differential expression of genes contributing to PCD triggered by exogenous oxalic acid in tomato (Solanum lycopersicum)

The present study aims at detecting Programmed Cell Death (PCD) related genes that were triggered by exogenous oxalic acid (OA) in tomato (Solanum lycopersicum L.). Reverse transcription polymerase chain reaction (RT-PCR) for the relative abundance of transcripts of four PCD-related genes in wild type (WT) plants resulted in increased expression levels during the time course of the treatment. The results of virus induced gene silencing (VIGS) lines corresponding to the two genes MEK2 and SGT1 retarded cell death under OA treatment for 24 h, while VIGS corresponding to the other two genes, e.g. BAK1 and plastocyanin resulted in an increased level of cell death. These results indicate that genes MEK2 and SGT1 showed apoptotic activity, while genes BAK1 and plastocyanin showed anti-apoptotic activity. It is speculated that knocking down MEK2 and SGT1 genes or over-expressing BAK1 and plastocyanin might be a possible way to retard the PCD machinery in tomato, thus can result in prolonged fruit setting time.

Modification of Potato Steroidal Glycoalkaloids with Silencing RNA Constructs

Steroidal glycoalkaloids (SGAs), while found in many solanaceous plants, can accumulate to unacceptably high levels in potato tubers. The two primary SGAs that occur in potatoes are the tri-glycosylated alkaloids, α-solanine and α-chaconine. The first glycosylation steps in their biosynthetic pathways are performed by the regulated enzymes SGT1, the UDP-galactose:solanidine galactosyltransferase, and SGT2, the UDP-glucose:solanidine glucosyltransferase, respectively. Using fragments of the Sgt1 and Sgt2 genes to produce small inhibitory RNAs (siRNA), we have been able to down-regulate each branch of the pathway. The use of the siRNA approach increases the efficiency of producing transgenic plant lines with reductions in individual SGAs but further research is required to achieve reductions in levels of total SGA accumulation.

Root-mediated signal transmission of systemic acquired resistance against above-ground and below-ground pathogens.

Background and Aims Plants modulate defence signalling networks in response to various biotic stresses via inter-organ communications. The root-mediated transmission of systemic acquired resistance (SAR) against soil-borne and air-borne plant pathogens from SAR-induced plants to neighbouring plants subjected to local chemical and pathogen treatments was evaluated. Methods The first two plants out of ten Nicotiana benthamiana seedlings were pre-treated with the SAR-triggering chemical benzothiadiazole (BTH). All ten seedlings were then challenged with two pathogenic bacteria, i.e. the root (bacterial wilt) pathogen Ralstonia solanacearum and the leaf (wildfire) pathogen Pseudomonas syringae pv. tabaci, at 7 d after SAR induction. Key Results Disease severity was noticeably lower in BTH-pre-treated plants than in the control. Surprisingly, two plants located next to BTH-treated plants exhibited reduced disease symptoms indicating that SAR signal transmission occurred through the root system. Determinant(s) secreted from the root system were search for and it was found that salicylic acid (SA) is a major molecule involved in SAR transmission through the root. Analysis of the expression of the defence-related genes N. benthamiana pathogenesis-related gene 1a (NbPR1a) and NbPR2 confirmed that BTH treatment elicited SAR via root-root transmission between plants. Plants with knock-down of the multiple resistance component SGT1 and SA biosynthesis-related gene ICS1 by Tobacco rattle virus-mediated virus-induced gene silencing exhibited a lack of root-mediated SAR transmission. The biological relevance of this finding was validated by challenge with the SAR-inducing avirulent pathogen P. syringae pv. syringae instead of BTH, which produced similar results. Conclusions Our findings demonstrated that SAR is transmissible through the root system from SAR-triggered plants to neighbouring plants.

Characterization and abiotic stress-responsive expression analysis of SGT1 genes in Brassica oleracea.

SGT1 genes are involved in enhancing plant responses to various biotic and abiotic stresses. Brassica oleracea is known to contain two types of SGT1 genes, namely suppressor of G2 allele of SKP1 and suppressor of GCR2. In this study, through systematic analysis, four putative SGT1 genes were identified and characterized in B. oleracea. In phylogenetic analysis, the genes clearly formed separate groups, namely BolSGT1a, BolSGT1b (both suppressor of G2 allele of SKP1 types), and BolSGT1 (suppressor of GCR2). Functional domain analysis and organ-specific expression patterns suggested possible roles for BolSGT1 genes during stress conditions. BolSGT1 genes showed significant changes in expression in response to heat, cold, drought, salt, or ABA treatment. Interaction network analysis supported the expression analysis, and showed that the BolSGT1a and BolSGT1b genes are strongly associated with co-regulators during stress conditions. However, the BolSGT1 gene did not show any strong association. Hence, BolSGT1 might be a stress resistance-related gene that functions without a co-regulator. Our results show that BolSGT1 genes are potential target genes to improve B. oleracea resistance to abiotic stresses such as heat, cold, and salt.

Characterization and Transcriptional Profile of Genes Involved in Glycoalkaloid Biosynthesis in New Varieties of Solanum tuberosum L.

Before commercial release, new potato (Solanum tuberosum) varieties must be evaluated for content of toxic compounds such as glycoalkaloids (GAs), which are potent poisons. GA biosynthesis proceeds via the cholesterol pathway to α-chaconine and α-solanine. The goal of this study was to evaluate the relationship between total glycoalkaloid (TGA) content and the expression of GAME, SGT1, and SGT3 genes in potato tubers. TGA content was measured by HPLC-MS, and reverse transcription quantitative polymerase chain reactions were performed to determine the relative expression of GAME, SGT1, and SGT3 genes. We searched for cis-elements of the transcription start site using the PlantPAN database. There was a relationship between TGA content and the relative expression of GAME, SGT1, and SGT3 genes in potato tubers. Putative promoter regions showed the presence of several cis-elements related to biotic and abiotic stresses and light. These findings provide an important step toward understanding TGA regulation and variation in potato tubers.

Impact of light-exposure on the metabolite balance of transgenic potato tubers with modified glycoalkaloid biosynthesis

Metabolite profiling (liquid chromatography–mass spectrometry (LC–MS) and gas chromatography (GC–MS)) was used to assess the impact of light on the composition of transgenic potato (Solanum tuberosum L. cv. Desirée) with reduced glycoalkaloid content via the down-regulation of the SGT1 gene. Transgenic tubers exhibited an almost complete knock-out of α-solanine production and light had little impact on its accumulation. Levels of α-chaconine increased significantly in the peel of both the control and transgenic lines when exposed to light, particularly in the transgenic line. Major differences in metabolite profiles existed between outer and inner tuber tissues, and between light and dark-treated tubers. Many of the light-induced changes are explicable in terms of pathways known to be affected by stress responses. The impact of transgenesis on profiles was much less than that of tissue type or light and most differences were explicable in terms of the modification to the glycoalkaloid pathway.

Tomato plant cell death induced by inhibition of HSP90 is alleviated by Tomato yellow leaf curl virus infection.

To ensure a successful long-term infection cycle, begomoviruses must restrain their destructive effect on host cells and prevent drastic plant responses, at least in the early stages of infection. The monopartite begomovirus Tomato yellow leaf curl virus (TYLCV) does not induce a hypersensitive response and cell death on whitefly-mediated infection of virus-susceptible tomato plants until diseased tomatoes become senescent. The way in which begomoviruses evade plant defences and interfere with cell death pathways is still poorly understood. We show that the chaperone HSP90 (heat shock protein 90) and its co-chaperone SGT1 (suppressor of the G2 allele of Skp1) are involved in the establishment of TYLCV infection. Inactivation of HSP90, as well as silencing of the Hsp90 and Sgt1 genes, leads to the accumulation of damaged ubiquitinated proteins and to a cell death phenotype. These effects are relieved under TYLCV infection. HSP90-dependent inactivation of 26S proteasome degradation and the transcriptional activation of the heat shock transcription factors HsfA2 and HsfB1 and of the downstream genes Hsp17 and Apx1/2 are suppressed in TYLCV-infected tomatoes. Following suppression of the plant stress response, TYLCV can replicate and accumulate in a permissive environment.

Identification and Characterization of Tomato Mutants Affected in theRx-Mediated Resistance to PVX Isolates

Five tomato mutants affected in the Rx-mediated resistance against Potato virus X (PVX) were identified by screening a mutagenized population derived from a transgenic, Rx1-expressing 'Micro-Tom' line. Contrary to their parental line, they failed to develop lethal systemic necrosis upon infection with the virulent PVX-KH2 isolate. Sequence analysis and quantitative reverse-transcription polymerase chain reaction experiments indicated that the mutants are not affected in the Rx1 transgene or in the Hsp90, RanGap1 and RanGap2, Rar1 and Sgt1 genes. Inoculation with the PVX-CP4 avirulent isolate demonstrated that the Rx1 resistance was still effective in the mutants. In contrast, the virulent PVX-KH2 isolate accumulation was readily detectable in all mutants, which could further be separated in two groups depending on their ability to restrict the accumulation of PVX-RR, a mutant affected at two key positions for Rx1 elicitor activity. Finally, transient expression of the viral capsid protein elicitor indicated that the various mutants have retained the ability to mount an Rx1-mediated hypersensitive response. Taken together, the results obtained are consistent with a modification of the specificity or intensity of the Rx1-mediated response. The five Micro-Tom mutants should provide very valuable resources for the identification of novel tomato genes affecting the functioning of the Rx gene.

One‐step, zero‐background ligation‐independent cloning intron‐containing hairpin RNA constructs for RNAi in plants

*The hairpin-based RNA interference (RNAi) technique plays an important role in exploring gene function in plants. Although there are several methods for making hairpin RNA (hpRNA) constructs, these methods usually need multiple relatively laborious, time-consuming or high-cost cloning steps. Here we describe a one-step, zero-background ligation-independent cloning (OZ-LIC) method for making intron-containing hpRNA (ihpRNA) constructs by our vector pRNAi-LIC. *To generate the ihpRNA constructs with zero-background, this method only requires treating two PCR products of target gene flanked with different LIC sequences and SmaI-linearized pRNAi-LIC vector by T4 DNA polymerase respectively, and then transforming these treated DNA mixture into Escherichia coli. *The ihpRNA constructs generated with our OZ-LIC RNAi vector can efficiently induce not only transient silencing of the exogenous marker genes and the endogenous resistance-related Nicotiana benthamiana SGT1 gene, but also stable transgenic suppression of Arabidopsis SGT1b gene. *Our new OZ-LIC method and RNAi vector will represent a powerful tool for gene knockdown in plants and may facilitate high-throughput determination of plant gene function.

Abstract A78: Sgt1 heterozygous mutation suppresses phenotypes of p53-null mice

Abstract Sgt1 is an Hsp90 co-chaperone that is highly conserved among species, from yeast to humans. In vitro studies using siRNA and small-molecule inhibitors have indicated that Sgt1 is required for kinetochore assembly, proper localization of the spindle checkpoint proteins during mitosis, and genomic stability in humans. In vivo studies using knockout (KO) mice have revealed that kinetochore and spindle checkpoint genes are related to tumorigenesis. To examine the in vivo functions of Sgt1 gene in mammals, especially in tumorigenesis, we generated and analyzed Sgt1 KO mice by using a gene-trap line. No Sgt1 homozygous KO mice were obtained from heterozygous intercrosses, even in E8.5 embryos, suggesting that Sgt1 homozygous mice are early embryonic lethal. In contrast, Sgt1 heterozygous mutants grew normally and did not exhibit any obvious phenotypes. We are now monitoring carcinogen-induced and spontaneous tumorigenesis in Sgt1 heterozygous mutants. We generated Sgt1 p53 double mutants in order to enhance the phenotypes associated with tumorigenesis. p53 is a major tumor suppressor gene, and p53 −/− KO mice usually die when they are approximately 20 weeks old, mainly because of lymphoma. Interestingly, Sgt1+/− p53−/− double mutants, especially females, survive significantly longer than p53−/− single mutants, suggesting that the Sgt1 heterozygous mutation suppresses p53−/− -induced tumorigenesis. In addition to the survival phenotype, the unequal gender ratio (more males than females) in p53−/− mutants was also overcome by the Sgt1 heterozygous mutation. These results suggest that the Sgt1 heterozygous mutation suppresses the p53 KO phenotypes not only in tumorigenesis but also in embryonic development defects of females. We are currently examining whether the female infertility in p53−/− is also suppressed by the Sgt1 mutation. We will discuss how Sgt1 suppresses the p53 phenotypes at the meeting. Citation Information: Cancer Res 2009;69(23 Suppl):A78.

Arabidopsis Sgt1a as an important factor for the acquirement of thermotolerance

We previously isolated a combination of high-light and heat-shock stress (HL + HS)-inducible genes, including a suppressor of the G2 allele of skp1 ( Sgt1a) from Arabidopsis. Arabidopsis contains two Sgt1 genes ( Sgt1a and Sgt1b). Here we explored the physiological function of Arabidopsis Sgt1a or Sgt1b under stressful conditions. The transcript and protein levels of Sgt1a markedly increased under not only HL + HS stress, but also HS stress or H 2O 2 treatment. There was no significant difference in acquired thermotolerance between the wild-type, Sgt1a-overexpressing or Sgt1b-overexpressing plants or Sgt1b-disrupted mutants ( edm1). However, the acquired thermotolerance was lower in the knockout Sgt1a mutants (KO- Sgt1a) than the wild-type plants. Furthermore, the transcript levels of some heat shock proteins (Hsp) were significantly lower in the KO- Sgt1a mutants than the wild-type plants and edm1 mutants. The present findings indicated Sgt1a to be important for the acquired thermotolerance.

Evaluating the ability of the barley stripe mosaic virus-induced gene silencing system to simultaneously silence two wheat genes

Virus-induced gene silencing (VIGS) is an important tool for rapid assessment of gene function in plants. The ability of the Barley stripe mosaic virus (BSMV) VIGS system to simultaneously silence two genes was assessed by comparing the extent of down-regulation of the wheat PDS and SGT1 genes after wheat plants were infected with BSMV-VIGS constructs carrying 178bp fragments of the PDS and SGT1 genes linked in cis or after infection with single-gene silencing BSMV constructs carrying PDS or SGT1 alone or with a mixture of the single-gene constructs. Although significant variation occurred in these studies it was clearly demonstrated that two wheat genes can be silenced simultaneously by a single BSMV-VIGS construct.

Repression of the Auxin Response Pathway Increases Arabidopsis Susceptibility to Necrotrophic Fungi

In plants, resistance to necrotrophic pathogens depends on the interplay between different hormone systems, such as those regulated by salicylic acid (SA), jasmonic acid (JA), ethylene, and abscisic acid. Repression of auxin signaling by the SA pathway was recently shown to contribute to antibacterial resistance. Here, we demonstrate that Arabidopsis auxin signaling mutants axr1, axr2, and axr6 that have defects in the auxin-stimulated SCF (Skp1-Cullin-F-box) ubiquitination pathway exhibit increased susceptibility to the necrotrophic fungi Plectosphaerella cucumerina and Botrytis cinerea. Also, stabilization of the auxin transcriptional repressor AXR3 that is normally targeted for removal by the SCF-ubiquitin/proteasome machinery occurs upon P. cucumerina infection. Pharmacological inhibition of auxin transport or proteasome function each compromise necrotroph resistance of wild-type plants to a similar extent as in non-treated auxin response mutants. These results suggest that auxin signaling is important for resistance to the necrotrophic fungi P. cucumerina and B. cinerea. SGT1b (one of two Arabidopsis SGT1 genes encoding HSP90/HSC70 co-chaperones) promotes the functions of SCF E3-ubiquitin ligase complexes in auxin and JA responses and resistance conditioned by certain Resistance (R) genes to biotrophic pathogens. We find that sgt1b mutants are as resistant to P. cucumerina as wild-type plants. Conversely, auxin/SCF signaling mutants are uncompromised in RPP4-triggered resistance to the obligate biotrophic oomycete, Hyaloperonospora parasitica. Thus, the predominant action of SGT1b in R gene-conditioned resistance to oomycetes appears to be at a site other than assisting SCF E3-ubiquitin ligases. However, genetic additivity of sgt1b axr1 double mutants in susceptibility to H. parasitica suggests that SCF-mediated ubiquitination contributes to limiting biotrophic pathogen colonization once plant-pathogen compatibility is established.

Sgt1 has co-chaperone properties and is up-regulated by heat shock

The Sgt1 protein is a binding partner of heat shock proteins such as Hsp90, Hsp70 or Hsc70. In this work we show that the level of Sgt1 is increased in HEp-2 cells exposed to heat shock or radicicol. The citrate synthase aggregation assay shows that Sgt1 attenuates aggregation of the enzyme induced by increased temperature as efficiently as p23, a known co-chaperone of Hsp90. We have cloned two fragments of the human Sgt1 gene promoter (−708/+98 and −351/+98) into pGL3-luciferase vector and found that both fragments generated a 2-fold increase in luciferase activity upon heat shock. Furthermore, electrophoretic mobility shift assay revealed binding of the HSF-1 transcription factor to the heat shock element in the proximal (−42/−2) Sgt1 gene promoter fragment. These results indicate that Sgt1 is a co-chaperone protein with an expression pattern matching that of the well known heat shock proteins.

Sgt1, but not Rar1, is essential for the RB-mediated broad-spectrum resistance to potato late blight.

BackgroundLate blight is the most serious potato disease world-wide. The most effective and environmentally sound way for controlling late blight is to incorporate natural resistance into potato cultivars. Several late blight resistance genes have been cloned recently. However, there is almost no information available about the resistance pathways mediated by any of those genes.ResultsWe previously cloned a late blight resistance gene, RB, from a diploid wild potato species Solanum bulbocastanum. Transgenic potato lines containing a single RB gene showed a rate-limiting resistance against all known races of Phytophthora infestans, the late blight pathogen. To better understand the RB-mediated resistance we silenced the potato Rar1 and Sgt1 genes that have been implicated in mediating disease resistance responses against various plant pathogens and pests. The Rar1 and Sgt1 genes of a RB-containing potato clone were silenced using a RNA interference (RNAi)-based approach. All of the silenced potato plants displayed phenotypically normal growth. The late blight resistance of the Rar1 and Sgt1 silenced lines were evaluated by a traditional greenhouse inoculation method and quantified using a GFP-tagged P. infestans strain. The resistance of the Rar1-silenced plants was not affected. However, silencing of the Sgt1 gene abolished the RB-mediated resistance.ConclusionOur study shows that silencing of the Sgt1 gene in potato does not result in lethality. However, the Sgt1 gene is essential for the RB-mediated late blight resistance. In contrast, the Rar1 gene is not required for RB-mediated resistance. These results provide additional evidence for the universal role of the Sgt1 gene in various R gene-mediated plant defense responses.

The Mi-1-Mediated Pest Resistance Requires Hsp90 and Sgt1

The tomato (Solanum lycopersicum) Mi-1 gene encodes a protein with putative coiled-coil nucleotide-binding site and leucine-rich repeat motifs. Mi-1 confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphids (Macrosiphum euphorbiae), and sweet potato whitefly (Bemisia tabaci). To identify genes required in the Mi-1-mediated resistance to nematodes and aphids, we used tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) to repress candidate genes and assay for nematode and aphid resistance. We targeted Sgt1 (suppressor of G-two allele of Skp1), Rar1 (required for Mla12 resistance), and Hsp90 (heat shock protein 90), which are known to participate early in resistance gene signaling pathways. Two Arabidopsis (Arabidopsis thaliana) Sgt1 genes exist and one has been implicated in disease resistance. Thus far the sequence of only one Sgt1 ortholog is known in tomato. To design gene-specific VIGS constructs, we cloned a second tomato Sgt1 gene, Sgt1-2. The gene-specific VIGS construct TRV-SlSgt1-1 resulted in lethality, while silencing Sgt1-2 using TRV-SlSgt1-2 did not result in lethal phenotype. Aphid and root-knot nematode assays of Sgt1-2-silenced plants indicated no role for Sgt1-2 in Mi-1-mediated resistance. A Nicotiana benthamiana Sgt1 VIGS construct silencing both Sgt1-1 and Sgt1-2 yielded live plants and identified a role for Sgt1 in Mi-1-mediated aphid resistance. Silencing of Rar1 did not affect Mi-1-mediated nematode and aphid resistance and demonstrated that Rar1 is not required for Mi-1 resistance. Silencing Hsp90-1 resulted in attenuation of Mi-1-mediated aphid and nematode resistance and indicated a role for Hsp90-1. The requirement for Sgt1 and Hsp90-1 in Mi-1-mediated resistance provides further evidence for common components in early resistance gene defense signaling against diverse pathogens and pests.

The primary in vivo steroidal alkaloid glucosyltransferase from potato

To provide tools for breeders to control the steroidal glycoalkaloid (SGA) pathway in potato, we have investigated the steroidal alkaloid glycosyltransferase ( Sgt) gene family. The committed step in the SGA pathway is the glycosylation of solanidine by either UDP-glucose or UDP-galactose leading to α-chaconine or α-solanine, respectively. The Sgt2 gene was identified by deduced protein sequence homology to the previously identified Sgt1 gene. SGT1 has glucosyltransferase activity in vitro, but in vivo serves as the UDP-galactose:solanidine galactosyltransferase. Two alleles of the Sgt2 gene were isolated and its function was established with antisense transgenic lines and in vitro assays of recombinant protein. In tubers of transgenic potato ( Solanum tuberosum) cvs. Lenape and Desirée expressing an antisense Sgt2 gene construct, accumulation of α-solanine was increased and α-chaconine was reduced. Studies with recombinant SGT2 protein purified from yeast show that SGT2 glycosylation activity is highly specific for UDP-glucose as a sugar donor. This data establishes the function of the gene product (SGT2), as the primary UDP-glucose:solanidine glucosyltransferase in vivo.