Potato miR394 targets StA/N-INVE and StLCR to negatively regulate late blight resistance.

MicroRNAs (miRNAs) are small noncoding RNAs in eukaryotes. Plant endogenous miRNAs play pivotal roles in regulating plant development and defense responses. MicroRNA394 (miR394) has been reported to regulate plant development, abiotic stresses and defense responses. Previous reports showed that miR394 responded to P. infestans inoculation in potato, indicating that miR394 may be involved in defense responses. In this study, we further investigated its role in potato defense against P. infestans. Stable expression of miR394 in tobacco and potato enhances the susceptibility to P. infestans, which is accompanied with the reduced accumulation of ROS and down-regulation of the PTI (pattern-triggered immunity) marker genes. Besides well-known target StLCR, miR394 also targets StA/N-INVE, which encodes a chloroplast Alkaline/Neutral Invertases (A/N-INVE). Both StLCR and StA/N-INVE positively regulate late blight resistance, while miR394 degrades them. Interestingly, StA/N-INVE is located in the chloroplast, indicating that miR394 may manipulate chloroplast immunity. Degradation of StA/N-INVE may affect the chloroplast function and hence lead to the compromised ROS (reactive oxygen species) burst and reduced retrograde signaling from the chloroplast to the nucleus and cytoplasm. In summary, this study provides new information that miR394 targets and degrades StA/N-INVE and StLCR, which are positive regulators, to enhance potato susceptibility to P. infestans.

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.

A Phytophthora effector promotes homodimerization of host transcription factor StKNOX3 to enhance susceptibility.

Oomycete pathogens secrete hundreds of cytoplasmic RxLR effectors to modulate host immunity by targeting diverse plant proteins. Revealing how effectors manipulate host proteins is pivotal to understanding infection processes and to developing new strategies to control plant disease. Here we show that the Phytophthora infestans RxLR effector Pi22798 interacts in the nucleus with a potato class II knotted-like homeobox (KNOX) transcription factor, StKNOX3. Silencing the ortholog NbKNOX3 in Nicotiana benthamiana reduces host colonization by P. infestans, whereas transient and stable overexpression of StKNOX3 enhances infection. StKNOX3 forms a homodimer which is dependent on its KNOX II domain. The KNOX II domain is also essential for Pi22798 interaction and for StKNOX3 to enhance P. infestans colonization, indicating that StKNOX3 homodimerization contributes to susceptibility. However, critically, the effector Pi22798 promotes StKNOX3 homodimerization, rather than heterodimerization to another KNOX transcription factor StKNOX7. These results demonstrate that the oomycete effector Pi22798 increases pathogenicity by promoting homodimerization specifically of StKNOX3 to enhance susceptibility.

Phytophthora infestans RXLR effector Pi04089 perturbs diverse defense-related genes to suppress host immunity.

BACKGROUND: The oomycete pathogen secretes many effectors into host cells to manipulate host defenses. For the majority of effectors, the mechanisms related to how they alter the expression of host genes and reprogram defenses are not well understood. In order to investigate the molecular mechanisms governing the influence that the Phytophthora infestans RXLR effector Pi04089 has on host immunity, a comparative transcriptome analysis was conducted on Pi04089 stable transgenic and wild-type potato plants. RESULTS: Potato plants stably expressing Pi04089 were more susceptible to P. infestans. RNA-seq analysis revealed that 658 upregulated genes and 722 downregulated genes were characterized in Pi04089 transgenic lines. A large number of genes involved in the biological process, including many defense-related genes and certain genes that respond to salicylic acid, were suppressed. Moreover, the comparative transcriptome analysis revealed that Pi04089 significantly inhibited the expression of many flg22 (a microbe-associated molecular pattern, PAMP)-inducible genes, including various Avr9/Cf-9 rapidly elicited (ACRE) genes. Four selected differentially expressed genes (StWAT1, StCEVI57, StKTI1, and StP450) were confirmed to be involved in host resistance against P. infestans when they were transiently expressed in Nicotiana benthamiana. CONCLUSION: The P. infestans effector Pi04089 was shown to suppress the expression of many resistance-related genes in potato plants. Moreover, Pi04089 was found to significantly suppress flg22-triggered defense signaling in potato plants. This research provides new insights into how an oomycete effector perturbs host immune responses at the transcriptome level.

Evolutionarily distinct resistance proteins detect a pathogen effector through its association with different host targets.

Knowledge of the evolutionary processes which govern pathogen recognition is critical to understanding durable disease resistance. We determined how Phytophthora infestans effector PiAVR2 is recognised by evolutionarily distinct resistance proteins R2 and Rpi-mcq1. We employed yeast two-hybrid, co-immunoprecipitation, virus-induced gene silencing, transient overexpression, and phosphatase activity assays to investigate the contributions of BSL phosphatases to R2- and Rpi-mcq1-mediated hypersensitive response (R2 HR and Rpi-mcq1 HR, respectively). Silencing PiAVR2 target BSL1 compromises R2 HR. Rpi-mcq1 HR is compromised only when BSL2 and BSL3 are silenced. BSL1 overexpression increases R2 HR and compromises Rpi-mcq1. However, overexpression of BSL2 or BSL3 enhances Rpi-mcq1 and compromises R2 HR. Okadaic acid, which inhibits BSL phosphatase activity, suppresses both recognition events. Moreover, expression of a BSL1 phosphatase-dead (PD) mutant suppresses R2 HR, whereas BSL2-PD and BSL3-PD mutants suppress Rpi-mcq1 HR. R2 interacts with BSL1 in the presence of PiAVR2, but not with BSL2 and BSL3, whereas no interactions were detected between Rpi-mcq1 and BSLs. Thus, BSL1 activity and association with R2 determine recognition of PiAVR2 by R2, whereas BSL2 and BSL3 mediate Rpi-mcq1 perception of PiAVR2. R2 and Rpi-mcq1 utilise distinct mechanisms to detect PiAVR2 based on association with different BSLs, highlighting central roles of these effector targets for both disease and disease resistance.

The oomycete microbe-associated molecular pattern Pep-13 triggers SERK3/BAK1-independent plant immunity.

KEY MESSAGE: Oomycetes MAMP Pep-13 can trigger SERK3/BAK1-independent PTI. Silencing of SERK3/BAK1 in solanaceous plants resulted in reduced expression of brassinosteroid marker genes and enhanced PTI transcriptional responses to Pep-13 treatment. To prevent disease, pattern recognition receptors (PRRs) are responsible for detecting microbe-associated molecular patterns (MAMPs) to switch on plant innate immunity. SOMATIC EMBROYOGENESIS KINASE 3 (SERK3)/BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) is a well-characterized receptor-like kinase (RLK) that serves as a pivotal co-receptor with PRRs to activate immunity following recognition of MAMPs including flg22, EF-Tu, INF1 and XEG1. However, the requirement for SERK3/BAK1 in many pattern-triggered immune (PTI) signaling pathways is not yet known. Pep-13 is an oomycete MAMP that consists of a highly conserved motif (an oligopeptide of 13 amino acids) shared in Phytophthora transglutaminases. Quantitative RT-PCR analysis reveals that the transcripts of three PTI marker genes (WRKY7, WRKY8 and ACRE31) rapidly accumulate in response to three different MAMPs: flg22, chitin and Pep-13. Whereas silencing of SERK3/BAK1 in Nicotiana benthamiana or potato compromised transcript accumulation in response to flg22, it did not attenuate WRKY7, WRKY8 and ACRE31 up-regulation in response to chitin or Pep-13. This indicates that Pep-13 triggers immunity in a SERK3/BAK1-independent manner, similar to chitin. Surprisingly, silencing of SERK3/BAK1 led to significantly increased accumulation of PTI marker gene transcripts following Pep-13 or chitin treatment, compared to controls. This was accompanied by reduced expression of brassinosteroid (BR) marker genes StSTDH, StEXP8 and StCAB50 and StCHL1, which is a negative regulator of PTI, supporting previous reports that SERK3/BAK1-dependent BR signaling attenuates plant immunity. We provide Pep-13 as an alternative to chitin as a trigger of SERK3/BAK1-independent immunity.

Phytophthora infestans effector SFI3 targets potato UBK to suppress early immune transcriptional responses.

The potato blight agent Phytophthora infestans secretes a range of RXLR effectors to promote disease. Recent evidence indicates that some effectors suppress early pattern-triggered immunity (PTI) following perception of microbe-associated molecular patterns (MAMPs). Phytophthora infestans effector PiSFI3/Pi06087/PexRD16 has been previously shown to suppress MAMP-triggered pFRK1-Luciferase reporter gene activity. How PiSFI3 suppresses immunity is unknown. We employed yeast-two-hybrid (Y2H) assays, co-immunoprecipitation, transcriptional silencing by RNA interference and virus-induced gene silencing (VIGS), and X-ray crystallography for structure-guided mutagenesis, to investigate the function of PiSFI3 in targeting a plant U-box-kinase protein (StUBK) to suppress immunity. We discovered that PiSFI3 is active in the host nucleus and interacts in yeast and in planta with StUBK. UBK is a positive regulator of specific PTI pathways in both potato and Nicotiana benthamiana. Importantly, it contributes to early transcriptional responses that are suppressed by PiSFI3. PiSFI3 forms an unusual trans-homodimer. Mutation to disrupt dimerization prevents nucleolar localisation of PiSFI3 and attenuates both its interaction with StUBK and its ability to enhance P. infestans leaf colonisation. PiSFI3 is a 'WY-domain' RXLR effector that forms a novel trans-homodimer which is required for its ability to suppress PTI via interaction with the U-box-kinase protein StUBK.

A potato STRUBBELIG-RECEPTOR FAMILY member, StLRPK1, associates with StSERK3A/BAK1 and activates immunity.

Plant STRUBBELIG (SUB)-RECEPTOR FAMILY (SRF) genes encode putative leucine-rich repeat transmembrane receptor-like kinases. SRFs have been reported to play essential roles in tissue morphogenesis in many plant organs. Here, we show that a potato SRF family gene, StLRPK1, is involved in plant immunity. StLRPK1 is located at the cell plasma membrane and is strongly induced by culture filtrate from in vitro growth of the late blight pathogen Phytophthora infestans. Overexpression of StLRPK1 in stable transgenic potato or ectopic expression in Nicotiana benthamiana plants enhances P. infestans disease resistance, whereas RNA interference (RNAi) of StLRPK1 in potato decreases disease resistance. We found that StLRPK1 constitutively interacts with a pivotal co-receptor, SERK3A/BAK1, which plays a central role in plant immunity. Virus-induced gene silencing of SERK3A/BAK1 in N. benthamiana lines expressing StLRPK1 attenuated P. infestans resistance, indicating that SERK3A/BAK1 is required for StLRPK1-mediated immunity. Finally, we show that StLRPK1-triggered late blight resistance depends on the mitogen-activated protein kinase kinase MEK2 and mitogen-activated protein kinase WIPK. We propose a model in which StLRPK1 associates with SERK3A/BAK1 to positively regulate plant immunity to P. infestans through a MAPK cascade. These data provide new insights into our understanding of SRF function in plant immunity.

PAMP-responsive ATL gene StRFP1 and its orthologue NbATL60 positively regulate Phytophthora infestans resistance in potato and Nicotiana benthamiana.

Ubiquitination is a post-translational modification that plays a crucial role during the regulation of plant immune signalling. The plant ATL family consists of a large number of putative RING type ubiquitin ligases. We show that potato ATL family gene StRFP1 and its orthologue NbATL60 from N. benthamiana both respond to Phytophthora infestans culture filtrate (CF) and flg22 induction. StRFP1 positively regulates immunity against P. infestans in potato. Ectopic transient expression of StRFP1 or expression of NbATL60 in N. benthamiana also enhances late blight resistance. By contrast, silencing NbATL60 in N. benthamiana reduces late blight resistance and leads to plant growth inhibition. Both StRFP1 and NbATL60 localize to the plasma membrane and intracellular puncta and possess E3 Ligase activity in vitro. Furthermore we demonstrate that the RING finger domain mutants of StRFP1 and NbATL60 lost E3 ligase activity and fail to suppress P. infestans colonization in N. benthamiana, indicating that E3 ligase activity is critical for StRFP1 and NbATL60 to regulate immunity. Overexpression or RNA interference of StRFP1 in transgenic potato led to increased or decreased expression of PTI maker genes (WRKY7, WRKY8, ACRE31 and Pti5) respectively. Similarly silencing of NbATL60 in N. benthamiana decreases expression of these PTI marker genes. Moreover, VIGS of NbATL60 in N. benthamiana did not compromise P. infestans PAMP INF1 or R2/Avr2, R3a/AVR3a, Rx/Cp and Pto/AvrPto triggered cell death. These results indicate that ATL genes StRFP1 and NbATL60 contribute to basal immunity (PTI) in Solanaceous plants.

The Potato MAP3K StVIK Is Required for the Phytophthora infestans RXLR Effector Pi17316 to Promote Disease.

Plant pathogens deliver effectors to manipulate processes in their hosts, creating a suitable environment for invasion and proliferation. Yet, little is known about the host proteins that are targeted by effectors from filamentous pathogens. Here, we show that stable transgenic expression in potato (Solanum tuberosum) and transient expression in Nicotiana benthamiana of the arginine-any amino acid-leucine-arginine effector Pi17316 enhances leaf colonization by the late blight pathogen Phytophthora infestans Expression of Pi17316 also attenuates cell death triggered by the pathogen-associated molecular pattern Infestin1 (INF1), indicating that the effector suppresses pattern-triggered immunity. However, this effector does not attenuate cell death triggered by a range of resistance proteins, showing that it specifically suppresses INF1-triggered cell death (ICD). In yeast two-hybrid assays, Pi17316 interacts directly with the potato ortholog of VASCULAR HIGHWAY1-interacting kinase (StVIK), encoding a predicted MEK kinase (MAP3K). Interaction in planta was confirmed by coimmunoprecipitation and occurs at the plant plasma membrane. Virus-induced gene silencing of VIK in N. benthamiana attenuated P. infestans colonization, whereas transient overexpression of StVIK enhanced colonization, indicating that this host protein acts as a susceptibility factor. Moreover, VIK overexpression specifically attenuated ICD, indicating that it is a negative regulator of immunity. The abilities of Pi17316 to enhance P. infestans colonization or suppress ICD were compromised significantly in NbVIK-silenced plants, demonstrating that the effector activity of Pi17316 is mediated by this MAP3K. Thus, StVIK is exploited by P. infestans as a susceptibility factor to promote late blight disease.

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.

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.

Potato NPH3/RPT2-Like Protein StNRL1, Targeted by a Phytophthora infestans RXLR Effector, Is a Susceptibility Factor.

Plant pathogens deliver effectors to manipulate host processes. We know little about how fungal and oomycete effectors target host proteins to promote susceptibility, yet such knowledge is vital to understand crop disease. We show that either transient expression in Nicotiana benthamiana, or stable transgenic expression in potato (Solanum tuberosum), of the Phytophthora infestans RXLR effector Pi02860 enhances leaf colonization by the pathogen. Expression of Pi02860 also attenuates cell death triggered by the P. infestans microbe-associated molecular pattern INF1, indicating that the effector suppresses pattern-triggered immunity. However, the effector does not attenuate cell death triggered by Cf4/Avr4 coexpression, showing that it does not suppress all cell death activated by cell surface receptors. Pi02860 interacts in yeast two-hybrid assays with potato NPH3/RPT2-LIKE1 (NRL1), a predicted CULLIN3-associated ubiquitin E3 ligase. Interaction of Pi02860 in planta was confirmed by coimmunoprecipitation and bimolecular fluorescence complementation assays. Virus-induced gene silencing of NRL1 in N. benthamiana resulted in reduced P. infestans colonization and accelerated INF1-mediated cell death, indicating that this host protein acts as a negative regulator of immunity. Moreover, whereas NRL1 virus-induced gene silencing had no effect on the ability of the P. infestans effector Avr3a to suppress INF1-mediated cell death, such suppression by Pi02860 was significantly attenuated, indicating that this activity of Pi02860 is mediated by NRL1. Transient overexpression of NRL1 resulted in the suppression of INF1-mediated cell death and enhanced P. infestans leaf colonization, demonstrating that NRL1 acts as a susceptibility factor to promote late blight disease.

A Phytophthora infestans RXLR effector targets plant PP1c isoforms that promote late blight disease.

Plant pathogens deliver effectors to alter host processes. Knowledge of how effectors target and manipulate host proteins is critical to understand crop disease. Here, we show that in planta expression of the RXLR effector Pi04314 enhances leaf colonization by Phytophthora infestans via activity in the host nucleus and attenuates induction of jasmonic and salicylic acid-responsive genes. Pi04314 interacts with three host protein phosphatase 1 catalytic (PP1c) isoforms, causing their re-localization from the nucleolus to the nucleoplasm. Re-localization of PP1c-1 also occurs during infection and is dependent on an R/KVxF motif in the effector. Silencing the PP1c isoforms or overexpression of a phosphatase-dead PP1c-1 mutant attenuates infection, demonstrating that host PP1c activity is required for disease. Moreover, expression of PP1c-1mut abolishes enhanced leaf colonization mediated by in planta Pi04314 expression. We argue that PP1c isoforms are susceptibility factors forming holoenzymes with Pi04314 to promote late blight disease.

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.

The Potato ERF Transcription Factor StERF3 Negatively Regulates Resistance to Phytophthora infestans and Salt Tolerance in Potato.

Ethylene response factors (ERFs) are unique to the plant kingdom and play crucial roles in plant response to various biotic and abiotic stresses. We show here that a potato StERF3, which contains an ERF-associated amphiphilic repression (EAR) motif in its C-terminal region, negatively regulates resistance to Phytophthora infestans and salt tolerance in potato. The StERF3 promoter responds to induction by salicylic acid, ABA ethylene and NaCl, as well as P. infestans, the causal agent of potato late blight disease. StERF3 could bind to the GCC box element of the HIS3 promoter and activate transcription of HIS3 in yeast cells. Importantly, silencing of StERF3 in potato produced an enhanced foliage resistance to P. infestans and elevated plant tolerance to NaCl stress accompanied by the activation of defense-related genes (PR1, NPR1 and WRKY1). In contrast, StERF3-overexpressing plants showed reduced expression of these defense-related genes and enhanced susceptibility to P. infestans, suggesting that StERF3 functions as a negative regulator of downstream defense- and/or stress-related genes in potato. StERF3 is localized to the nucleus. Interestingly, yeast two-hybrid assay and a bimolecular fluorescence complementation (BiFC) test clarified that StERF3 could interact with other proteins in the cytoplasm which may lead to its re-localization between the nucleus and cytoplasm, revealing a novel means of StERF3 regulation. Taken together, these data provide new insights into the mechanism underlying how StERF3 negatively regulates late blight resistance and abiotic tolerance in potato and may have a potential use in engineering late blight resistance in potato.

The dihydrolipoyl acyltransferase gene BCE2 participates in basal resistance against Phytophthora infestans in potato and Nicotiana benthamiana.

Dihydrolipoyl acyltransferase (EC 2.3.1.12), a branched-chain α-ketoacid dehydrogenase E2 subunit (BCE2), catalyzes the transfer of the acyl group from the lipoyl moiety to coenzyme A. However, the role of BCE2 responding to biotic stress in plant is not clear. In this study, we cloned and characterized a BCE2 gene from potato, namely StBCE2, which was previously suggested to be involved in Phytophthora infestans-potato interaction. We found that the expression of StBCE2 was strongly induced by both P. infestans isolate HB09-14-2 and salicylic acid. Besides, when the homolog of StBCE2 in Nicotiana benthamiana named NbBCE2 was silenced, plants showed increased susceptibility to P. infestans and reduced accumulation of hydrogen peroxide (H2O2). Furthermore, we found that a marker gene NbrbohB involved in the production of reactive oxygen species, was also suppressed in NbBCE2-silenced plants. However, silencing of NbBCE2 had no significant effect on the hypersensitive responses trigged by INF1, R3a-AVR3a(KI) pair or Rpi-vnt1.1-AVR-vnt1.1 pair. Our results suggest that BCE2 is associated with the basal resistance to P. infestans by regulating H2O2 production.

Functional analysis of potato genes involved in quantitative resistance to Phytophthora infestans.

The most significant threat to potato production worldwide is the late blight disease, which is caused by the oomycete pathogen Phytophthora infestans. Based on previous cDNA microarrays and cDNA-amplified fragment length polymorphism analysis, 63 candidate genes that are expected to contribute to developing a durable resistance to late blight were selected for further functional analysis. We performed virus-induced gene silencing (VIGS) to these candidate genes on both Nicotiana benthamiana and potato, subsequently inoculated detached leaves and assessed the resistance level. Ten genes decreased the resistance to P. infestans after VIGS treatment. Among those, a lipoxygenase (LOX; EC 1.13.11.12) and a suberization-associated anionic peroxidase affected the resistance in both N. benthamiana and potato. Our results identify genes that may play a role in quantitative resistance mechanisms to late blight.