A highly sensitive biotin-based probe for small RNA northern blot and its application in dissecting miRNA function in pepper.

Small RNAs play important roles in regulation of plant development and response to various stresses. Northern blot is an important technique in small RNA research. Isotope- and biotin- (or digoxigenin) labeled probes are frequently used in small RNA northern blot. However, isotope-based probe is limited by strict environmental regulation and availability in many places in the world while biotin-based probe is usually suffered from low sensitivity. In this study, we developed a T4 DNA polymerase-based method for incorporation of a cluster of 33 biotin-labeled C in small RNA probe (T4BC33 probe). T4BC33 probe reaches similar sensitivity as 32P-labeled probe in dot blot and small RNA northern blot experiments. Addition of locked nucleic acids in T4BC33 probe further enhanced its sensitivity in detecting low-abundance miRNAs. With newly developed northern blot method, expression of miR6027 and miR6149 family members was validated. Northern blot analysis also confirmed the successful application of virus-based miRNA silencing in pepper, knocking down accumulation of Can-miR6027a and Can-miR6149L. Importantly, further analysis showed that knocking-down Can-miR6027a led to upregulation of a nucleotide binding-leucine rich repeat domain protein coding gene (CaRLb1) and increased immunity against Phytophthora capsici in pepper leaves. Our study provided a highly sensitive and convenient method for sRNA research and identified new targets for genetic improvement of pepper immunity against P. capsici.

Potato ß-aminobutyric acid receptor IBI1 manipulates VOZ1 and VOZ2 transcription factor activity to promote disease resistance.

Upon infection with non-pathogenic microorganisms or treatment with natural or synthetic compounds, plants exhibit a more rapid and potent response to both biotic and abiotic stresses. However, the molecular mechanisms behind this phenomenon, known as defense priming, are poorly understood. ß-aminobutyric acid (BABA) is an endogenous stress metabolite that enhances plant tolerance to various abiotic stresses and primes plant defense responses, providing the ability to resist a variety of pathogens (broad-spectrum resistance). In this study, we identified an aspartyl-tRNA synthetase (AspRS), StIBI1 (named after Arabidopsis  IMPAIRED IN BABA-INDUCED IMMUNITY 1; IBI1), as a BABA receptor in Solanum tuberosum. We elucidated the regulatory mechanisms by which StIBI1 interacts with two NAC (NAM, ATAF1, 2, and CUC2) transcription factors (TFs), StVOZ1 and StVOZ2 (VASCULAR PLANT ONE ZINC FINGER, VOZ), to activate BABA-induced resistance (BABA-IR). StVOZ1 represses, whereas StVOZ2 promotes, immunity to the late blight pathogen Phytophthora infestans. Interestingly, BABA and StIBI1 influence StVOZ1- and StVOZ2-mediated immunity. StIBI1 interacts with StVOZ1 and StVOZ2 in the cytoplasm, reducing the nuclear accumulation of StVOZ1 and promoting the nuclear accumulation of StVOZ2. Our findings indicate that StVOZ1 and StVOZ2 finely regulate potato resistance to late blight through distinct signaling pathways. In summary, our study provides insights into the interaction between the potato BABA receptor StIBI1 and the TFs StVOZ1 and StVOZ2, which affects StVOZ1 and StVOZ2stability and nuclear accumulation to regulate late blight resistance during BABA-IR. This research advances our understanding of the primary mechanisms of BABA-IR in potato and contributes to a theoretical basis for the prevention and control of potato late blight using BABA-IR.

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 Catalyst Tube-Equipped Dual-Stage Tube Furnace System for Accurate Hg Isotopic Determination of Ore Samples Using Neptune Plus Multicollector Inductively Coupled Plasma Mass Spectrometry.

Mercury (Hg) isotopes, which display mass-dependent fractionation and mass-independent fractionation, provide a multidimensional tracer to decipher the source of metals in mineral deposits. However, mineral ore samples usually contain abundant interfering elements (e.g., Te) that can cause inaccurate Hg isotopic analysis. Available acid digestion and combustion methods failed to remove these interfering elements, hindering the application of Hg isotopes for metallogenetic tracing. Here, we developed a new dual-stage tube furnace system employing a Mn-containing catalyst tube to pretreat mineral ore samples. This method yielded good Hg recoveries (100.5 ± 3.8%, 1SD, n = 15) and low levels of interfering elements in sample solutions, allowing for accurate analysis of a series of ore standard reference materials (GBW-11108v: coal; GSO-3: Cu-Ag sulfide ore; GBW 07859: Au-Te sulfide ore). The new method was also successfully applied to measure the Hg isotopic composition of magmatic and hydrothermal ore deposits, which yielded a large range in Δ199Hg value (-0.19 to 0.22‰) for ore deposits formed in different geological settings, highlighting the future applications of this method for metallogenic tracing, especially tracing the source of metals in mineral ore deposits.

Potato E3 ubiquitin ligase StRFP1 positively regulates late blight resistance by degrading sugar transporters StSWEET10c and StSWEET11.

Potato (Solanum tuberosum) is the fourth largest food crop in the world. Late blight, caused by oomycete Phytophthora infestans, is the most devastating disease threatening potato production. Previous research has shown that StRFP1, a potato Arabidopsis Tóxicos en Levadura (ATL) family protein, positively regulates late blight resistance via its E3 ligase activity. However, the underlying mechanism is unknown. Here, we reveal that StRFP1 is associated with the plasma membrane (PM) and undergoes constitutive endocytic trafficking. Its PM localization is essential for inhibiting P. infestans colonization. Through in vivo and in vitro assays, we investigated that StRFP1 interacts with two sugar transporters StSWEET10c and StSWEET11 at the PM. Overexpression (OE) of StSWEET10c or StSWEET11 enhances P. infestans colonization. Both StSWEET10c and StSWEET11 exhibit sucrose transport ability in yeast, and OE of StSWEET10c leads to an increased sucrose content in the apoplastic fluid of potato leaves. StRFP1 ubiquitinates StSWEET10c and StSWEET11 to promote their degradation. We illustrate a novel mechanism by which a potato ATL protein enhances disease resistance by degrading susceptibility (S) factors, such as Sugars Will Eventually be Exported Transporters (SWEETs). This offers a potential strategy for improving disease resistance by utilizing host positive immune regulators to neutralize S factors.

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.

Genetic variations in MdSAUR36 participate in the negative regulation of mesocarp cell division and fruit size in Malus species.

Final fruit size of apple (Malus domestica) cultivars is related to both mesocarp cell division and cell expansion during fruit growth, but it is unclear whether the cell division and/or cell enlargement determine most of the differences in fruit size between Malus species. In this study, by using an interspecific hybrid population between Malus asiatica "Zisai Pearl" and Malus domestica cultivar "Red Fuji," we found that the mesocarp cell number was the main causal factor of diversity in fruit size between Malus species. Rapid increase in mesocarp cell number occurred prior to 28 days after anthesis (DAA), while cell size increased gradually after 28 DAA until fruit ripening. Six candidate genes related to auxin signaling or cell cycle were predicted by combining the RNA-seq data and previous QTL data for fruit weight. Two InDels and 10 SNPs in the promoter of a small auxin upregulated RNA gene MdSAUR36 in Zisai Pearl led to a lower promoter activity than that of Red Fuji. One non-synonymous SNP G/T at 379 bp downstream of the ATG codon of MdSAUR36, which was heterozygous in Zisai Pearl, exerted significant genotype effects on fruit weight, length, and width. Transgenic apple calli by over-expressing or RNAi MdSAUR36 confirmed that MdSAUR36 participated in the negative regulation of mesocarp cell division and thus apple fruit size. These results could provide new insights in the molecular mechanism of small fruit size in Malus accession and be potentially used in molecular assisted breeding via interspecific hybridization. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01441-4.

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 dihydroflavonol 4-reductase BoDFR1 drives anthocyanin accumulation in pink-leaved ornamental kale.

KEY MESSAGE: Overexpression and virus-induced gene silencing verified BoDFR1 conferred the anthocyanin accumulation in pink-leaved ornamental kale. Leaf color is an essential trait in the important horticultural biennial plant ornamental kale (Brassica oleracea var. acephala). The identity of the gene conferring this striking trait and its mode of inheritance are topics of debate. Based on an analysis of F1, F2, BC1P1, and BC1P2 ornamental kale populations derived from a cross between a pink-leaved P28 and white-leaved D10 line, we determined that the pink leaf trait is controlled by a semi-dominant gene. We cloned two genes potentially involved in anthocyanin biosynthesis in ornamental kale: Bo9g058630 and Bo6g100940. Based on their variation in sequence, we speculated that Bo9g058630, encoding the kale dihydroflavonol-4 reductase (BoDFR1) enzyme, plays a critical role in the development of the pink leaf trait. Indeed, an InDel marker specific for BoDFR1 completely co-segregated with the pink leaf trait in our F2 population. We then generated the 35Spro: DFR-GUS overexpression vector, which we transformed into D10. Overexpression of BoDFR1 indeed restored some anthocyanin accumulation in this white-leaved parental line. In addition, we targeted BoDFR1 in P28 using virus-induced gene silencing. Again, silencing of BoDFR1 resulted in a substantial decrease in anthocyanin accumulation. This work lays the foundation for further exploration of the mechanism underlying anthocyanin accumulation in pink-leaved ornamental kale.

Phytophthora infestans RXLR Effectors Target Parallel Steps in an Immune Signal Transduction Pathway.

The potato (Solanum tuberosum) blight pathogen Phytophthora infestans delivers Arg-X-Leu-Arg (RXLR) effector proteins into host cells to subvert plant immune responses and promote colonization. We show that transient expression and stable transgenic expression of the RXLR effector Pi22926 in Nicotiana benthamiana promotes leaf colonization by P. infestans. Pi22926 suppresses cell death triggered by coexpression of the Cladosporium fulvum avirulence protein Avr4 and the tomato (Solanum lycopersicum) resistance protein Cf4. Pi22926 interacts with a potato mitogen-activated protein kinase kinase kinase, StMAP3Kß2, in the nucleoplasm. Virus-induced gene silencing (VIGS) of the ortholog NbMAP3Kß2 in N. benthamiana enhances P. infestans colonization and attenuates Cf4/Avr4-induced cell death, indicating that this host protein is a positive regulator of immunity. Cell death induced by Cf4/Avr4 is dependent on NbMAP3Kε and NbMAP3Kß2, indicating that these MAP3Ks function in the same signaling pathway. VIGS of NbMAP3Kß2 does not compromise cell death triggered by overexpression of MAP3Kε. Similarly, VIGS of NbMAP3Kε does not attenuate cell death triggered by MAP3Kß2, demonstrating that these MAP3K proteins function in parallel. In agreement, Pi22926 or another RXLR effector, PexRD2, only suppresses cell death triggered by expression of StMAP3Kß2 or StMAP3Kε, respectively. Our data reveal that two P. infestans effectors, PexRD2 and Pi22926, promote P. infestans colonization by targeting MAP3K proteins that act in parallel in the same signal transduction pathway.

BTB-BACK Domain Protein POB1 Suppresses Immune Cell Death by Targeting Ubiquitin E3 ligase PUB17 for Degradation.

Hypersensitive response programmed cell death (HR-PCD) is a critical feature in plant immunity required for pathogen restriction and prevention of disease development. The precise control of this process is paramount to cell survival and an effective immune response. The discovery of new components that function to suppress HR-PCD will be instrumental in understanding the regulation of this fundamental mechanism. Here we report the identification and characterisation of a BTB domain E3 ligase protein, POB1, that functions to suppress HR-PCD triggered by evolutionarily diverse pathogens. Nicotiana benthamiana and tobacco plants with reduced POB1 activity show accelerated HR-PCD whilst those with increased POB1 levels show attenuated HR-PCD. We demonstrate that POB1 dimerization and nuclear localization are vital for its function in HR-PCD suppression. Using protein-protein interaction assays, we identify the Plant U-Box E3 ligase PUB17, a well established positive regulator of plant innate immunity, as a target for POB1-mediated proteasomal degradation. Using confocal imaging and in planta immunoprecipitation assays we show that POB1 interacts with PUB17 in the nucleus and stimulates its degradation. Mutated versions of POB1 that show reduced interaction with PUB17 fail to suppress HR-PCD, indicating that POB1-mediated degradation of PUB17 U-box E3 ligase is an important step for negative regulation of specific immune pathways in plants. Our data reveals a new mechanism for BTB domain proteins in suppressing HR-PCD in plant innate immune responses.

Hair, encoding a single C2H2 zinc-finger protein, regulates multicellular trichome formation in tomato.

Trichomes originate from the epidermal cells of nearly all terrestrial plants, which are specialized unicellular or multicellular structures. Although the molecular mechanism regulating unicellular trichome formation has been extensively characterized, most of the genes essential for multicellular trichome formation remain unknown. In this study, we identified an associated locus on the long arm of chromosome 10 using a genome-wide association study (GWAS) on type-I trichomes of 180 diverse Solanum lycopersicum (tomato) accessions. Using map-based cloning we then cloned the key gene controlling the initiation of this type of trichome, named Hair (H), which encodes a single C2H2 zinc-finger protein. Transgenic experiments showed that hair-absent phenotype is caused by the deletion of the entire coding region of H. We identified three alleles of H containing several missense mutations and a nucleotide deletion, which result in amino acid substitutions and a reading frame shift, respectively. In addition, knockdown of H or Woolly (Wo) represses the formation of type-I trichomes, suggesting that both regulators may function as a heterodimer. Direct protein-protein interaction between them was further detected through pull-down and yeast two-hybrid assays. In addition, ectopic expression of H in Nicotiana tabacum (tobacco) and expression of its homologs from Capsicum annuum (pepper) and tobacco in tomato can trigger trichome formation. Taken together, these findings suggest that the H gene may be functionally conserved in multicellular trichome formation in Solanaceae species.

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.

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.

Phytophthora infestans RXLR effectors act in concert at diverse subcellular locations to enhance host colonization.

Oomycetes such as the potato blight pathogen Phytophthora infestans deliver RXLR effectors into plant cells to manipulate host processes and promote disease. Knowledge of where they localize inside host cells is important in understanding their function. Fifty-two P. infestans RXLR effectors (PiRXLRs) up-regulated during early stages of infection were expressed as fluorescent protein (FP) fusions inside cells of the model host Nicotiana benthamiana. FP-PiRXLR fusions were predominantly nucleo-cytoplasmic, nuclear, or plasma membrane-associated. Some also localized to the endoplasmic reticulum, mitochondria, peroxisomes, or microtubules, suggesting diverse sites of subcellular activity. Seven of the 25 PiRXLRs examined during infection accumulated at sites of haustorium penetration, probably due to co-localization with host target processes; Pi16663 (Avr1), for example, localized to Sec5-associated mobile bodies which showed perihaustorial accumulation. Forty-five FP-RXLR fusions enhanced pathogen leaf colonization when expressed in Nicotiana benthamiana, revealing that their presence was beneficial to infection. Co-expression of PiRXLRs that target and suppress different immune pathways resulted in an additive enhancement of colonization, indicating the potential to study effector combinations using transient expression assays. We provide a broad platform of high confidence P. infestans effector candidates from which to investigate the mechanisms, singly and in combination, by which this pathogen causes disease.

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.

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.

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.