A Phytophthora infestans RXLR effector AVR8 suppresses plant immunity by targeting a desumoylating isopeptidase DeSI2.

The potato's most devastating disease is late blight, which is caused by Phytophthora infestans. Whereas various resistance (R) genes are known, most are typically defeated by this fast-evolving oomycete pathogen. However, the broad-spectrum and durable R8 is a vital gene resource for potato resistance breeding. To support an educated deployment of R8, we embarked on a study on the corresponding avirulence gene Avr8. We overexpressed Avr8 by transient and stable transformation, and found that Avr8 promotes colonization of P. infestans in Nicotiana benthamiana and potato, respectively. A yeast-two-hybrid (Y2H) screen showed that AVR8 interacts with a desumoylating isopeptidase (StDeSI2) of potato. We overexpressed DeSI2 and found that DeSI2 positively regulates resistance to P. infestans, while silencing StDeSI2 downregulated the expression of a set of defense-related genes. By using a specific proteasome inhibitor, we found that AVR8 destabilized StDeSI2 through the 26S proteasome and attenuated early PTI responses. Altogether, these results indicate that AVR8 manipulates desumoylation, which is a new strategy that adds to the plethora of mechanisms that Phytophthora exploits to modulate host immunity, and StDeSI2 provides a new target for durable resistance breeding against P. infestans in potato.

Rychc Confers Extreme Resistance to Potato virus Y in Potato.

The Potato virus Y (PVY) is responsible for huge economic losses for the potato industry worldwide and is the fifth most consequential plant virus globally. The main strategies for virus control are to limit aphid vectors, produce virus-free seed potatoes, and breed virus-resistant varieties. The breeding of PVY-resistant varieties is the safest and most effective method in terms of cost and environmental protection. Rychc, a gene that confers extreme resistance to PVY, is from S. chacoense, which is a wild diploid potato species that is widely used in many PVY-resistant breeding projects. In this study, Rychc was fine mapped and successfully cloned from S. chacoense accession 40-3. We demonstrated that Rychc encodes a TIR-NLR protein by stably transforming a diploid susceptible cultivar named AC142 and a tetraploid potato variety named E3. The Rychc conferred extreme resistance to PVYO, PVYN:O and PVYNTN in both of the genotypes. To investigate the genetic events occurring during the evolution of the Rychc locus, we sequenced 160 Rychc homologs from 13 S. chacoense genotypes. Based on the pattern of sequence identities, 160 Rychc homologs were divided into 11 families. In Family 11 including Rychc, we found evidence for Type I evolutionary patterns with frequent sequence exchanges, obscured orthologous relationships and high non-synonymous to synonymous substitutions (Ka/Ks), which is consistent with rapid diversification and positive selection in response to rapid changes in the PVY genomes. Furthermore, a functional marker named MG64-17 was developed in this study that indicates the phenotype with 100% accuracy and, therefore, can be used for marker-assisted selection in breeding programs that use S. chacoense as a breeding resource.

Global Screening and Functional Identification of Major HSPs Involved in PVY Infection in Potato.

HSP40 (also known as DnaJ), HSP70, and HSP90 are major heat shock protein (HSP) families that play critical roles in plant growth and development and stress adaption. Recently, several members of the three HSP families were reported to be widely involved in the plant host-virus interactions. However, their global expression profiles and core members recruited by viruses are largely unknown. In this study, a total of 89 StDnaJs were identified from a genome-wide survey, and their classification, phylogenetic relationships, chromosomal locations, and gene duplication events were further analyzed. Together with 20 StHSP70s and 7 StHSP90s previously identified in the potato genome, the global expression patterns of the members in 3 HSP families were investigated in 2 potato cultivars during Potato virus Y (PVY) infection using RNA-seq data. Of them, 16 genes (including 8 StDnaJs, 6 StHSP70s, and 2 StHSP90s) were significantly up- or downregulated. Further analysis using qRT-PCR demonstrated that 7 of the 16 genes (StDnaJ06, StDnaJ17, StDnaJ21, StDnaJ63, StHSP70-6, StHSP70-19, and StHSP90.5) were remarkably upregulated in the potato cultivar 'Eshu 3' after PVY infection, implying their potential roles in the potato-PVY compatible interaction. Subsequent virus-induced gene silencing (VIGS) assays showed that silencing of the homologous genes of StDnaJ17, StDnaJ21, StHSP70-6, and StHSP90.5 in Nicotiana. benthamiana plants dramatically reduced the accumulation of PVY, which indicated the four genes may function as susceptibility factors in PVY infection. This study provides candidate genes for exploring the mechanism of potato-PVY compatible interaction and benefits breeding work aiming to produce new cultivars with the ability to grow healthily under PVY infection.

Extreme Resistance to Potato Virus A in Potato Cultivar Barbara is Independently Mediated by Ra and Rysto.

Potato virus A (PVA) and potato virus Y (PVY) are two members of genus Potyvirus infecting potato crops worldwide. Host resistance offers an economical and effective means for the control or management of these viruses. In this study, 20 potato clones were screened for their resistance against PVA and PVY by mechanical or graft inoculation assay, and were explored for the relationship between extreme resistance genes Ra and Ry by the detection of molecular markers linked to Ryadg, Rysto, and Rychc. Six clones, including Barbara, Jizhangshu 8, Longshu 7, Longshu 8, M6, and Solara, were found to be extremely resistant to both PVA and PVY; three clones (AC142, Eshu 3, and Shepody) were deemed to be extremely resistant to PVA but susceptible to PVY. To further reveal the inheritance of the extreme resistance (ER) against PVA, a tetraploid F1 population of Barbara × F58050 (susceptible to both PVY and PVA) and a tetraploid BC1 population of BF145 (a PVA-resistant but PVY-susceptible progeny of Barbara × F58050) × F58050 were obtained. Phenotyping of the F1 and BC1 populations by graft inoculation with PVA showed segregation ratios of 3:1 and 1:1 (resistant:susceptible), respectively. These results suggest that two independent loci control ER against PVA in Barbara: one confers ER to both PVA and PVY and the other confers ER to PVA only. The deduced genotype of Barbara is RyryryryRararara.

Comparative transcriptome reveals distinct starch-sugar interconversion patterns in potato genotypes contrasting for cold-induced sweetening capacity.

Potato accumulates large amounts of soluble sugar during cold storage periods. However, a system based understanding of this process is still largely unknown. Here, we compared the dynamic cold-responded transcriptome of genotypes between cold-induced sweetening resistant (CIS-R) and cold-induced sweetening sensitive (CIS-S) in tubers. Comparative transcriptome revealed that activating the pathways of starch degradation, sucrose synthesis and hydrolysis could be common strategies in response to cold in both genotypes. Moreover, the variation in sugar accumulation between genotypes may be due to genetic differences in cold response, which could be mainly explained: CIS-R genotype was active in starch synthesis and attenuated in sucrose hydrolysis by promoting the coordinate expression of aseries ofgenes involved in starch-sugar interconversion. Additionally, transcription factors, the candidate master regulators of starch-sugar interconversion, were discussed. Taken together, this work has provided an avenue for studying the mechanism involved in the regulation of the CIS resistance.

SbRFP1 regulates cold-induced sweetening of potato tubers by inactivation of StBAM1.

Potato cold-induced sweetening (CIS) is a major drawback restricting potato process industry. Starch degradation and sucrose decomposition are considered to be the key pathways in potato CIS. Our previous study showed that the RING finger gene SbRFP1 could slow down starch degradation and the accumulation of reducing sugars (RS) through inhibiting amylase and invertase activity in cold-stored tubers. However, the regulation mechanism of SbRFP1 is not clear. In this paper, we first proved that SbRFP1 could promote starch synthesis and modify the shape of starch granules. By further yeast two hybrid, GST-pull down and inhibition of enzyme activity assays, we confirmed that SbRFP1 could slow down the transformation of starch to RS in tubers mainly through the inhibition of ß-amylase StBAM1 activity. SbRFP1 was also proved to possess E3 ubiquitin ligase activity by ubiquitination assay. Thus, SbRFP1 may regulate the accumulation of RS in cold-stored tubers by ubiquitination and degradation of StBAM1. Therefore, our study reveals the regulatory mechanism of SbRFP1 in the process of CIS and provides more powerful evidence for the effect of starch degradation on potato CIS.

A systematic screen of conserved Ralstonia solanacearum effectors reveals the role of RipAB, a nuclear-localized effector that suppresses immune responses in potato.

Both Solanum tuberosum and Ralstonia solanacearum phylotype IIB originated in South America and share a long-term co-evolutionary history. However, our knowledge of potato bacterial wilt pathogenesis is scarce as a result of the technical difficulties of potato plant manipulation. Thus, we established a multiple screening system (virulence screen of effector mutants in potato, growth inhibition of yeast and transient expression in Nicotiana benthamiana) of core type III effectors (T3Es) of a major potato pathovar of phylotype IIB, to provide more research perspectives and biological tools. Using this system, we identified four effectors contributing to virulence during potato infection, with two exhibiting multiple phenotypes in two other systems, including RipAB. Further study showed that RipAB is an unknown protein with a nuclear localization signal (NLS). Furthermore, we generated a ripAB complementation strain and transgenic ripAB-expressing potato plants, and subsequent virulence assays confirmed that R. solanacearum requires RipAB for full virulence. Compared with wild-type potato, transcriptomic analysis of transgenic ripAB-expressing potato plants showed a significant down-regulation of Ca2+ signalling-related genes in the enriched Plant-Pathogen Interaction (PPI) gene ontology (GO) term. We further verified that, during infection, RipAB is required for the down-regulation of four Ca2+ sensors, Stcml5, Stcml23, Stcml-cast and Stcdpk2, and a Ca2+ transporter, Stcngc1. Further evidence showed that the immune-associated reactive oxygen species (ROS) burst is attenuated in ripAB transgenic potato plants. In conclusion, a systematic screen of conserved R. solanacearum effectors revealed an important role for RipAB, which interferes with Ca2+ -dependent gene expression to promote disease development in potato.

Phytochrome F plays critical roles in potato photoperiodic tuberization.

The transition to tuberization contributes greatly to the adaptability of potato to a wide range of environments. Phytochromes are important light receptors for the growth and development of plants, but the detailed functions of phytochromes remain unclear in potato. In this study, we first confirmed that phytochrome F (StPHYF) played essential roles in photoperiodic tuberization in potato. By suppressing the StPHYF gene, the strict short-day potato genotype exhibited normal tuber formation under long-day (LD) conditions, together with the degradation of the CONSTANTS protein StCOL1 and modulation of two FLOWERING LOCUS T (FT) paralogs, as demonstrated by the repression of StSP5G and by the activation of StSP6A during the light period. The function of StPHYF was further confirmed through grafting the scion of StPHYF-silenced lines, which induced the tuberization of untransformed stock under LDs, suggesting that StPHYF was involved in the production of mobile signals for tuberization in potato. We also identified that StPHYF exhibited substantial interaction with StPHYB both in vitro and in vivo. Therefore, our results indicate that StPHYF plays a role in potato photoperiodic tuberization, possibly by forming a heterodimer with StPHYB.

The arginine decarboxylase gene ADC1, associated to the putrescine pathway, plays an important role in potato cold-acclimated freezing tolerance as revealed by transcriptome and metabolome analyses.

Low temperature severely influences potato production as the cultivated potato (Solanum tuberosum) is frost sensitive, however the mechanism underlying the freezing tolerance of the potato is largely unknown. In the present research, we studied the transcriptome and metabolome of the freezing-tolerant wild species Solanum acaule (Aca) and freezing-sensitive cultivated S. tuberosum (Tub) to identify the main pathways and important factors related to freezing tolerance. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation indicated that polyamine and amino acid metabolic pathways were specifically upregulated in Aca under cold treatment. The transcriptome changes detected in Aca were accompanied by the specific accumulation of putrescine, saccharides, amino acids and other metabolites. The combination of transcriptome and metabolome analyses revealed that putrescine exhibited an accumulative pattern in accordance with the expression of the arginine decarboxylase gene ADC1. The primary role of putrescine was further confirmed by analyzing all three polyamines (putrescine, spermidine, and spermine) and the genes encoding the corresponding enzymes in two sets of potato genotypes with distinct freezing tolerance, implying that only putrescine and ADC1 were uniquely enhanced by cold in the freezing-tolerant genotypes. The function of putrescine was further analyzed by its exogenous application and the overexpression of SaADC1 in S. tuberosum cv. E3, indicating its important role(s) in cold-acclimated freezing tolerance, which was accompanied with the activation of C-repeat binding factor genes (CBFs). The present research has identified that the ADC1-associated putrescine pathway plays an important role in cold-acclimated freezing tolerance of potato, probably by enhancing the expression of CBF genes.

Radiosensitization of clioquinol and zinc in human cancer cell lines.

BACKGROUND: We previously reported that clioquinol acts as a zinc ionophore and inhibits the NF-κB signalling pathway. Other research has demonstrated that zinc deficiency plays a vital role in the occurrence and development of some solid tumours, and intracellular zinc supplementation may reverse this process and enhance the tumour sensitivity to anticancer treatment. Thus, we investigated the radiosensitization effects of clioquinol combined with zinc on HeLa and MCF-7 cells in vitro. METHODS: The dose effect of growth inhibition of clioquinol combined with zinc on cell viability was determined by a cell counting kit 8 (CCK-8) assay. The radiosensitization effect of clioquinol combined with zinc and/or MG132 in HeLa and MCF-7 cells was detected by the clonogenic assay. The cell cycle distribution and apoptosis of clioquinol combined with zinc on HeLa cells were analyzed by flow cytometry. A luciferase reporter construct was used to study the effect of clioquinol combined with zinc on NF-κB activity in HeLa cells. DNA double-strand breaks were detected by immunofluorescence. The mRNA and protein levels of ATM were analyzed by quantitative real-time PCR and Western blotting, respectively. RESULTS: Our research showed that clioquinol combined with zinc markedly increased the radiosensitivity of HeLa and MCF-7 cells in low toxic concentrations and resulted in a post-irradiation decrease in G2 phase arrest and an increase in apoptosis. Clioquinol combined with zinc also inhibited NF-κB activation, decreased ATM expression and increased DNA double-strand breaks (DSBs) induced by ionizing radiation. CONCLUSIONS: These findings indicated that clioquinol combined with zinc enhanced the radiosensitivity of HeLa and MCF-7 cells by the down-regulation of ATM through the NF-κB signalling pathway.

Potato late blight field resistance from QTL dPI09c is conferred by the NB-LRR gene R8.

Following the often short-lived protection that major nucleotide binding, leucine-rich-repeat (NB-LRR) resistance genes offer against the potato pathogen Phytophthora infestans, field resistance was thought to provide a more durable alternative to prevent late blight disease. We previously identified the QTL dPI09c on potato chromosome 9 as a more durable field resistance source against late blight. Here, the resistance QTL was fine-mapped to a 186 kb region. The interval corresponds to a larger, 389 kb, genomic region in the potato reference genome of Solanum tuberosum Group Phureja doubled monoploid clone DM1-3 (DM) and from which functional NB-LRRs R8, R9a, Rpi-moc1, and Rpi_vnt1 have arisen independently in wild species. dRenSeq analysis of parental clones alongside resistant and susceptible bulks of the segregating population B3C1HP showed full sequence representation of R8. This was independently validated using long-range PCR and screening of a bespoke bacterial artificial chromosome library. The latter enabled a comparative analysis of the sequence variation in this locus in diverse Solanaceae. We reveal for the first time that broad spectrum and durable field resistance against P. infestans is conferred by the NB-LRR gene R8, which is thought to provide narrow spectrum race-specific resistance.

StPOTHR1, a NDR1/HIN1-like gene in Solanum tuberosum, enhances resistance against Phytophthora infestans.

A family of NDR1/HIN1-like (NHL) genes that shows homology to the nonrace-specific disease resistance (NDR1) and the tobacco (Nicotiana tabacum) harpin-induced (HIN1) genes is reported to be involved in defense. However, little information about NHL genes is available for the potato (Solanum tuberosum). Here, we report that the expression of StPOTHR1, a member of the NHL gene family, is associated with resistance in potato against Phytophthora infestans, and is specifically induced in inoculation sites. Overexpression of StPOTHR1 enhances resistance against P. infestans via restricting rapid pathogen proliferation. Further, suppression of StPOTHR1 does not compromise R-mediated cell death. Subcellular localization and posttranscription modifications (PTMs) analysis reveals that StPOTHR1 is localized in plasma membrane (PM) and undergoes multiple PTMs. Moreover, StPOTHR1 interacts with NbMKK5L, a component of the MAP kinase signaling cascade. Taken together, our results suggest that the PM-localized StPOTHR1 contributes to potato immunity against P. infestans and may be associated with the MAP kinase signaling cascade.

Trichosanthin enhances sensitivity of non-small cell lung cancer (NSCLC) TRAIL-resistance cells.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has a specific antitumour activity against many malignant tumours. However, more than half of lung cancer cells are resistant to TRAIL-relevant drugs. Trichosanthin (TCS) is a traditional Chinese medicine with strong inhibitive effects on various malignancies. Nevertheless, its function on TRAIL resistance has not been revealed in non-small cell lung cancer (NSCLC). To examine the molecular mechanisms of TCS-induced TRAIL sensitivity, we administrated TCS to TRAIL-resistance NSCLC cells, and found that the combination treatment of TCS and TRAIL inhibited cancer cell proliferation and invasion, and induced cell apoptosis and S-phase arrest. This combined therapeutic method regulated the expression levels of extrinsic apoptosis-associated proteins Caspase 3/8 and PARP; intrinsic apoptosis-associated proteins BCL-2 and BAX; invasion-associated proteins E-cadherin, N-cadherin, Vimentin, ICAM-1, MMP-2 and MMP-9; and cell cycle-associated proteins P27, CCNE1 and CDK2. Up-expression and redistribution of death receptors (DRs) on the cell surface were also observed in combined treatment. In conclusion, our results indicated that TCS rendered NSCLC cells sensitivity to TRAIL via upregulating and redistributing DR4 and DR5, inducing apoptosis, and regulating invasion and cell cycle related proteins. Our results provided a potential therapeutic method to enhance TRAIL-sensitivity.

Cytosolic glyceraldehyde-3-phosphate dehydrogenases play crucial roles in controlling cold-induced sweetening and apical dominance of potato (Solanum tuberosum L.) tubers.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an important enzyme that functions in producing energy and supplying intermediates for cellular metabolism. Recent researches indicate that GAPDHs have multiple functions beside glycolysis. However, little information is available for functions of GAPDHs in potato. Here, we identified 4 putative cytosolic GAPDH genes in potato genome and demonstrated that the StGAPC1, StGAPC2, and StGAPC3, which are constitutively expressed in potato tissues and cold inducible in tubers, encode active cytosolic GAPDHs. Cosuppression of these 3 GAPC genes resulted in low tuber GAPDH activity, consequently the accumulation of reducing sugars in cold stored tubers by altering the tuber metabolite pool sizes favoring the sucrose pathway. Furthermore, GAPCs-silenced tubers exhibited a loss of apical dominance dependent on cell death of tuber apical bud meristem (TAB-meristem). It was also confirmed that StGAPC1, StGAPC2, and StGAPC3 interacted with the autophagy-related protein 3 (ATG3), implying that the occurrence of cell death in TAB-meristem could be induced by ATG3 associated events. Collectively, the present research evidences first that the GAPC genes play crucial roles in diverse physiological and developmental processes in potato tubers.

Amylases StAmy23, StBAM1 and StBAM9 regulate cold-induced sweetening of potato tubers in distinct ways.

Cold-induced sweetening (CIS) in potato is detrimental to the quality of processed products. Conversion of starch to reducing sugars (RS) by amylases is considered one of the main pathways in CIS but is not well studied. The amylase genes StAmy23, StBAM1, and StBAM9 were studied for their functions in potato CIS. StAmy23 is localized in the cytoplasm, whereas StBAM1 and StBAM9 are targeted to the plastid stroma and starch granules, respectively. Genetic transformation of these amylases in potatoes by RNA interference showed that ß-amylase activity could be decreased in cold-stored tubers by silencing of StBAM1 and collective silencing of StBAM1 and StBAM9. However, StBAM9 silencing did not decrease ß-amylase activity. Silencing StBAM1 and StBAM9 caused starch accumulation and lower RS, which was more evident in simultaneously silenced lines, suggesting functional redundancy. Soluble starch content increased in RNAi-StBAM1 lines but decreased in RNAi-StBAM9 lines, suggesting that StBAM1 may regulate CIS by hydrolysing soluble starch and StBAM9 by directly acting on starch granules. Moreover, StBAM9 interacted with StBAM1 on the starch granules. StAmy23 silencing resulted in higher phytoglycogen and lower RS accumulation in cold-stored tubers, implying that StAmy23 regulates CIS by degrading cytosolic phytoglycogen. Our findings suggest that StAmy23, StBAM1, and StBAM9 function in potato CIS with varying levels of impact.

D2-resected stage IIIc gastric cancer patients benefit from adjuvant chemoradiotherapy.

Although adjuvant chemoradiotherapy has been an important part in the treatment of gastric cancer, whether or not adjuvant radiation can benefit patients undergoing resection with D2 lymph node dissection remains controversial. This retrospective study aimed to evaluate the role of adjuvant chemoradiotherapy on patients with D2-resected gastric cancer. A total of 337 patients with resected gastric cancer treated at Zhongnan Hospital of Wuhan University from 2004 to 2012 were retrospectively analyzed. Eligible patients were divided into the adjuvant chemoradiotherapy group (CRT; n = 124) and the adjuvant chemotherapy group (CT; n = 213). The primary endpoints were disease-free survival (DFS) and overall survival (OS), with toxicity as the secondary endpoint. A subgroup analysis was performed based on clinical staging. The two groups were comparable in baseline characteristic, except for the number of lymph nodes dissected. The median OSs in the CRT and CT groups were 51.0 months and 48.6 months, respectively (P = 0.251), and the median DFSs were 40.7 months and 31.2 months, respectively (P = 0.112). Subgroup analysis revealed that the median OSs in patients at stage IIIc in the CRT group and CT group were 29.0 and 23.0 months, respectively (P = 0.049), and those of the median DFSs were 21.2 and 15.1 months, respectively (P = 0.015). There was no significant difference in main adverse events between two groups. Collectively, adjuvant chemoradiotherapy in gastric cancer patients with D2 resection was well tolerated. For Stage IIIc patients, the addition of adjuvant chemoradiotherapy was associated with a significant benefit in both OS and DFS.

Chinese expert consensus on cytoreductive surgery and hyperthermic intraperitoneal chemotherapy for peritoneal malignancies.

Locoregional spread of abdominopelvic malignant tumors frequently results in peritoneal carcinomatosis (PC). The prognosis of PC patients treated by conventional systemic chemotherapy is poor, with a median survival of < 6 mo. However, over the past three decades, an integrated treatment strategy of cytoreductive surgery (CRS) + hyperthermic intraperitoneal chemotherapy (HIPEC) has been developed by the pioneering oncologists, with proved efficacy and safety in selected patients. Supported by several lines of clinical evidence from phases I, II and III clinical trials, CRS + HIPEC has been regarded as the standard treatment for selected patients with PC in many established cancer centers worldwide. In China, an expert consensus on CRS + HIPEC has been reached by the leading surgical and medical oncologists, under the framework of the China Anti-Cancer Association. This expert consensus has summarized the progress in PC clinical studies and systematically evaluated the CRS + HIPEC procedures in China as well as across the world, so as to lay the foundation for formulating PC treatment guidelines specific to the national conditions of China.

Subtle Regulation of Potato Acid Invertase Activity by a Protein Complex of Invertase, Invertase Inhibitor, and SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE.

Slowing down cold-induced sweetening (CIS) of potato (Solanum tuberosum) tubers is of economic importance for the potato industry to ensure high-quality products. The conversion of sucrose to reducing sugars by the acid invertase StvacINV1 is thought to be critical for CIS. Identification of the specific StvacINV1 inhibitor StInvInh2B and the α- and ß-subunits of the interacting protein SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE from the wild potato species Solanum berthaultii (SbSnRK1) has led to speculation that invertase activity may be regulated via a posttranslational mechanism that remains to be elucidated. Using bimolecular fluorescence complementation assays, this study confirmed the protein complex by pairwise interactions. In vitro kinase assays and protein phosphorylation analysis revealed that phosphorylation of SbSnRK1α is causal for StvacINV1 activity and that its active form blocks the inhibition of StInvInh2B by SbSnRK1ß, whereas its inactive form restores the function of SbSnRK1ß that prevents StInvInh2B from repressing StvacINV1. Overexpression of SbSnRK1α in CIS-sensitive potato confirmed that SbSnRK1α has significant effects on acid invertase-associated sucrose degradation. A higher level of SbSnRK1α expression was accompanied by elevated SbSnRK1α phosphorylation, reduced acid invertase activity, a higher sucrose-hexose ratio, and improved chip color. Our results lend new insights into a subtle regulatory mode of invertase activity and provide a novel approach for potato CIS improvement.

Construction of efficient, tuber-specific, and cold-inducible promoters in potato.

Promoter activity is crucial for precise gene expression. Previously, a synthetic tuber-specific and cold-inducible promoter, pCL, containing a C-repeat/dehydration-responsive element (CRT/DRE) cassette and a tuber-specific fragment, was constructed in order to regulate cold-induced sweetening (CIS) in potatoes. However, the utility of pCL is limited due to its low activity. To improve its inducibility in response to low temperatures, we modified the CRT/DRE and flanking sequences. In particular, promoter activity was significantly improved by site-specific mutation of flanking sequences next to the core element (CCGAC) of CRT/DRE. We also inserted a modified CRT/DRE cassette into pCL; although this enhanced activity, it was not more effective than mutation of the flanking sequences. Indeed, up to 20-fold enhanced pCL activity could be achieved by replacing the CRT/DRE cassette in pCL with tandem repeats of two mutated CRT/DRE cassettes. This improvement was due to an enhanced affinity between the CRT/DRE cassette(s) and the StCBF1 transcription factor. Together, these data suggest that altering the structure of CRT/DRE can enhance CBF-related transcription complex formation and thus improve the activity of this cold-inducible promoter.

U-box E3 ubiquitin ligase PUB17 acts in the nucleus to promote specific immune pathways triggered by Phytophthora infestans.

Ubiquitination regulates many processes in plants, including immunity. The E3 ubiquitin ligase PUB17 is a positive regulator of programmed cell death (PCD) triggered by resistance proteins CF4/9 in tomato. Its role in immunity to the potato late blight pathogen, Phytophthora infestans, was investigated here. Silencing StPUB17 in potato by RNAi and NbPUB17 in Nicotiana benthamiana by virus-induced gene silencing (VIGS) each enhanced P. infestans leaf colonization. PAMP-triggered immunity (PTI) transcriptional responses activated by flg22, and CF4/Avr4-mediated PCD were attenuated by silencing PUB17. However, silencing PUB17 did not compromise PCD triggered by P. infestans PAMP INF1, or co-expression of R3a/AVR3a, demonstrating that not all PTI- and PCD-associated responses require PUB17. PUB17 localizes to the plant nucleus and especially in the nucleolus. Transient over-expression of a dominant-negative StPUB17(V314I,V316I) mutant, which retained nucleolar localization, suppressed CF4-mediated cell death and enhanced P. infestans colonization. Exclusion of the StPUB17(V314I,V316I) mutant from the nucleus abolished its dominant-negative activity, demonstrating that StPUB17 functions in the nucleus. PUB17 is a positive regulator of immunity to late blight that acts in the nucleus to promote specific PTI and PCD pathways.