The caffeoyl-CoA O-methyltransferase gene SNP replacement in Russet Burbank potato variety enhances late blight resistance through cell wall reinforcement.

KEY MESSAGE: Metabolic pathway gene editing in tetraploid potato enhanced resistance to late blight. Multiallelic mutation correction of a caffeoyl-CoA O-methyltransferase gene increased accumulation of resistance metabolites in Russet Burbank potato. Late blight of potato is a devastating disease worldwide and requires weekly applications of fungicides to manage. Genetic improvement is the best option, but the self-incompatibility and inter-specific incompatibility makes potato breeding very challenging. Immune receptor gene stacking has increased resistance, but its durability is limited. Quantitative resistance is durable, and it mainly involves secondary cell wall thickening due to several metabolites and their conjugates. Deleterious mutations in biosynthetic genes can hinder resistance metabolite biosynthesis. Here a probable resistance role of the StCCoAOMT gene was first confirmed by an in-planta transient overexpression of the functional StCCoAOMT allele in late blight susceptible Russet Burbank (RB) genotype. Following this, a precise single nucleotide polymorphism (SNP) mutation correction of the StCCoAOMT gene in RB potato was carried out using CRISPR-Cas9 mediated homology directed repair (HDR). The StCCoAOMT gene editing increased the transcript abundance of downstream biosynthetic resistance genes. Following pathogen inoculation, several phenylpropanoid pathway genes were highly expressed in the edited RB plants, as compared to the non-edited. The disease severity (fold change = 3.76) and pathogen biomass in inoculated stems of gene-edited RB significantly reduced (FC = 21.14), relative to non-edited control. The metabolic profiling revealed a significant increase in the accumulation of resistance-related metabolites in StCCoAOMT edited RB plants. Most of these metabolites are involved in suberization and lignification. The StCCoAOMT gene, if mutated, can be edited in other potato cultivars to enhance resistance to late blight, provided it is associated with other functional genes in the metabolic pathway network.

Identification and functional characterisation of late blight resistance polymorphic genes in Russet Burbank potato cultivar.

In plants, the biosynthesis of the phenylpropanoid, flavonoid and fatty acid pathway monomers, polymers and conjugated metabolites play a vital role in disease resistance. These are generally deposited to reinforce cell walls to contain the pathogen to the site of infection. Identification of sequence variants in genes that biosynthesise these resistance metabolites can explain the mechanisms of disease resistance. The resistant and susceptible genotypes inoculated with Phytophthora infestans were RNA sequenced to identify the single nucleotide polymorphisms (SNPs) and insertion/deletion (InDel) variations. The SNPs/InDels were annotated and classified into different categories based on their effect on gene functions. In the selected 25 biosynthetic genes overlapping 39 transcripts, a total of 52 SNPs/InDels were identified in the protein-coding (CDS) regions. These were categorised as deleterious based on prediction of their effects on protein structure and function. The SNPs/InDels data obtained in this study can be used in genome editing to enhance late blight resistance in Russet Burbank and other potato cultivars.

Genome-wide in silico identification of LysM-RLK genes in potato (Solanum tuberosum L.).

The receptor like kinases (RLKs) belong to the RLK/Pelle superfamily, one of the largest gene families in plants. RLKs play an important role in plant development, as well as in response to biotic and abiotic stresses. The lysine motif receptor like kinases (LysM-RLKs) are a subfamily of RLKs containing at least one lysine motif (LysM) that are involved in the perception of elicitors or pathogen-associated molecular patterns (PAMPs). In the present study, 77 putative RLKs genes and three receptor like proteins were identified in potato (Solanum tuberosum) genome, following a genome-wide search. The 77 potato RLK proteins are classified into two major phylogenetic groups based on their kinase domain amino acid sequence similarities. Out of 77 RLKs, 10 proteins had at least one LysM. Among them three RLP proteins were found in potato genome with either 2 or three tandem LysM but these lacked a cytoplasmic kinase domain. Expression analyses of a potato LysM-RLKs (StLysM-RLK05) was carried out by a Real time RT-PCR, following inoculation of potato leaves and immature tubers with late blight and common scab pathogens, respectively. The expression was significantly higher in resistant than in susceptible following S. scabies inoculation. The StLysM-RLK05 sequence was verified and it was polymorphic in scab susceptible cultivar. The present study provides an overview of the StLysM-RLKs gene family in potato genome. This information is helpful for future functional analysis of such an important protein family, in Solanaceae species.

StWRKY8 transcription factor regulates benzylisoquinoline alkaloid pathway in potato conferring resistance to late blight.

The resistance to late blight is either qualitative or quantitative in nature. Quantitative resistance is durable, but challenging due to polygenic inheritance. In the present study, the diploid potato genotypes resistant and susceptible to late blight, were profiled for metabolites. Tissue specific metabolite analysis of benzylisoquinoline alkaloids (BIAs) in response to pathogen infection revealed increased accumulation of morphinone, codeine-6-glucuronide and morphine-3-glucuronides. These BIAs are antimicrobial compounds and possibly involved in cell wall reinforcement, especially through cross-linking cell wall pectins. Quantitative reverse transcription-PCR studies revealed higher expressions of TyDC, NCS, COR-2 and StWRKY8 transcription factor genes, in resistant genotypes than in susceptible genotype, following pathogen inoculation. A luciferase transient expression assay confirmed the binding of the StWRKY8 TF to promoters of downstream genes, elucidating a direct regulatory role on BIAs biosynthetic genes. Sequence analysis of StWRKY8 in potato genotypes revealed polymorphism in the WRKY DNA binding domain in the susceptible genotype, which is important for the regulatory function of this gene. A complementation assay of StWRKY8 in Arabidopsis wrky33 mutant background was associated with decreased fungal biomass. In conclusion, StWRKY8 regulates the biosynthesis of BIAs that are both antimicrobial and reinforce cell walls to contain the pathogen to initial infection.

Natural variation of sucrose, glucose and fructose contents in Colombian genotypes of Solanum tuberosum Group Phureja at harvest.

BACKGROUND: Potato frying quality is a complex trait influenced by sugar content in tubers. Good frying quality requires low content of reducing sugars to avoid the formation of dark pigments. Solanum tuberosum Group Phureja is a valuable genetic resource for breeding and for genetic studies. The sugar content after harvest was analyzed in a germplasm collection of Group Phureja to contribute to the understanding of the natural variation of this trait. RESULTS: Sucrose, glucose and fructose genotypic mean values ranged from 6.39 to 29.48 g kg(-1) tuber dry weight (DW), from 0.46 to 28.04 g kg(-1) tuber DW and from 0.29 to 27.23 g kg(-1) tuber DW, respectively. Glucose/fructose and sucrose/reducing sugars ratios ranged from 1.01 to 6.67 mol mol(-1) and from 0.15 to 7.78 mol mol(-1) , respectively. Five clusters of genotypes were recognized, three of them with few genotypes and extreme phenotypic values. CONCLUSION: Sugar content showed a wide variation, representing the available variability useful for potato breeding. The results provide a quantitative approach to analyze the frying quality trait and are consistent with frying color. The analyzed germplasm presents extreme phenotypes, which will contribute to the understanding of the genetic basis of this trait. © 2016 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Transcription factor StWRKY1 regulates phenylpropanoid metabolites conferring late blight resistance in potato.

Quantitative resistance is polygenically controlled and durable, but the underlying molecular and biochemical mechanisms are poorly understood. Secondary cell wall thickening is a critical process in quantitative resistance, regulated by transcriptional networks. This paper provides compelling evidence on the functionality of StWRKY1 transcription factor, in a compatible interaction of potato-Phytophthora infestans, to extend our knowledge on the regulation of the metabolic pathway genes leading to strengthening the secondary cell wall. A metabolomics approach was used to identify resistance-related metabolites belonging to the phenylpropanoid pathway and their biosynthetic genes regulated by StWRKY1. The StWRKY1 gene in resistant potato was silenced to decipher its role in the regulation of phenylpropanoid pathway genes to strengthen the secondary cell wall. Sequencing of the promoter region of StWRKY1 in susceptible genotypes revealed the absence of heat shock elements (HSEs). Simultaneous induction of both the heat shock protein (sHSP17.8) and StWRKY1 following pathogen invasion enables functioning of the latter to interact with the HSE present in the resistant StWRKY1 promoter region. EMSA and luciferase transient expression assays further revealed direct binding of StWRKY1 to promoters of hydroxycinnamic acid amide (HCAA) biosynthetic genes encoding 4-coumarate:CoA ligase and tyramine hydroxycinnamoyl transferase. Silencing of the StWRKY1 gene was associated with signs of reduced late blight resistance by significantly increasing the pathogen biomass and decreasing the abundance of HCAAs. This study provides convincing evidence on the role of StWRKY1 in the regulation of downstream genes to biosynthesize HCAAs, which are deposited to reinforce secondary cell walls.

Development and validation of a liquid chromatographic method to quantify sucrose, glucose, and fructose in tubers of Solanum tuberosum Group Phureja.

A High Performance Liquid Chromatography (HPLC) method was developed and validated to quantify sucrose (non-reducing sugar), glucose, and fructose (reducing sugars) in raw tubers of Solanum tuberosum Group Phureja. Chromatographic analysis was performed using an AMINEX HPX 87H column, at 18 °C, linked to a refraction index detector, at 35 °C. The eluent was 10mM sulfuric acid. The conditions established for the method provided an optimum separation of sugars, citric acid, and malic acid, with resolution values higher or equal to one. Among the four sugar extraction methods tested, the double 50% (v/v) aqueous methanol extraction gave the highest level of analytes. Recovery of this extraction method ranged between 94.14 and 99.77%. The HPLC method was validated for repeatability, reproducibility, linearity, and limits of detection, and quantification. Relative standard deviation was found to be lower than five, when testing repeatability and reproducibility, which is suitable considering a range of acceptability from 5.3 to 7.3. Additionally, the regression analyses supported the method linearity in a range of quantification from 3 to 100 mg/L with regression coefficients values greater than 0.998 for the three analytes. Limits of detection were 3.0 mg/L for the three sugars and limits of quantification were 2.0 mg/L for sucrose and 3.0 mg/L for glucose and fructose. Four Colombian commercial cultivars (Criolla Guaneña, Criolla Paisa, Criolla Galeras, and Criolla Colombia) and five landrace accessions from the Colombian Core Collection of Group Phureja were grown in the district of Usme (Bogotá) fields to analyze their sugar contents. Sucrose, glucose, and fructose contents were found ranging from 0.93 to 3.11 g/100 g tuber dried weight (DW), from 0.25 to 4.53 g/100 g tuber DW, and from 0.10 to 1.49 g/100 g tuber DW, respectively. Therefore, a high range in the variability of sugar contents was found among genotypes. However, the variability was low among technical replicates of the same genotype, revealing an accurate quantification of sugars in Group Phureja. This method can be used to assess the amount of reducing and non-reducing sugars accumulation in potato germplasm.

Quantitative resistance in potato leaves to late blight associated with induced hydroxycinnamic acid amides.

Late blight is a serious economic threat to potato crop, sometimes leading to complete crop loss. The resistance in potato to late blight can be qualitative or quantitative in nature. Qualitative resistance is not durable. Though quantitative resistance is durable, the breeding is challenging due to polygenic inheritance. Several quantitative trait loci (QTLs) have been identified, but the mechanisms of resistance are largely unknown. A nontargeted metabolomics approach was used to identify resistance-related (RR) metabolites in a resistant genotype (F06025), as compared to a susceptible (Shepody) genotype, mock- or pathogen-inoculated. The RR metabolites, which had high fold change in abundance, mainly belonged to phenylpropanoid, flavonoid, fatty acid, and alkaloid chemical groups. The most important phenylpropanoids identified were hydroxycinnamic acid amides, the polyaromatic domain of suberin that is known to be associated with cell wall reinforcement. These metabolites were mapped on to the potato metabolic pathways, and the candidate enzymes and their coding genes were identified. A quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay revealed a higher upregulation of 4-coumarate: CoA ligase (4-CL), tyrosine decarboxylase (TyDC), and tyramine hydroxycinnamoyl transferase (THT) in the pathogen-inoculated resistant genotype than in susceptible. These genes were sequenced in both resistant and susceptible genotypes, and nonsynonymous single-nucleotide polymorphisms (nsSNPs) were found. The application of these genes in potato resistance improvement, following validation, is discussed.

Identification of metabolites related to mechanisms of resistance in barley against Fusarium graminearum, based on mass spectrometry.

Fusarium head blight (FHB) is an economically important disease of the family Triticeae, as, apart from yield reduction it also causes quality deterioration by producing mycotoxins. Host resistance is the most promising way to control the disease. Metabolic profiling was applied to identify resistance related (RR) metabolites against Fusarium graminearum in five FHB-resistant genotypes ('Chevron', 'H5277-44', 'H5277-164', 'M92-513' and 'M122') relative to one FHB-susceptible genotype ('Stander'). The disease severity was assessed in greenhouse to group the genotypes based on FHB-resistance. The disease was quantified as the proportion of diseased spikelets (PSD) and the area under the disease progress curve (AUDPC). Spikelets were collected at 72 h post inoculation. Metabolites were extracted into an aqueous solution of methanol and analyzed using a LC-hybrid-MS system. Metabolite abundances were subjected to a resistant versus susceptible pair-wise analysis, using a t test. Resistance related (RR) metabolites, both constitutive (RRC) and induced (RRI), were identified amongst metabolites whose levels were significantly higher in resistant genotype than in susceptible. Among 1,430 RR metabolites, 115 were putatively identified. These RR metabolites belonged to different chemical groups: fatty acids: linolenic acid; phenylpropanoids: p-coumaric, sinapic acid; flavonoids: naringenin, kaempferol glucoside, catechol glucoside. In addition, resistance indicator metabolites, such as deoxynivalenol (DON) and DON-3-O-glucoside (D3G) were also detected. The amount of total DON synthesized converted to D3G (PDC) was the greatest in resistant genotype 'Chevron' (PDC = 0.76). The role of the resistance-related and resistance-indicator metabolites on plant defense, and their use as potential biomarkers to screen barley genotypes for FHB resistance is discussed.

Efeito de extratos proteícos de milho sobre a produçäo de aflatoxina B / Effect of corn proteic extracts on aflatoxin B production

Testou-se o efeito de extratos proteícos de milho à base de NaC1 5% sobre a produçäo de aflatoxina B, em meio de cultura líquido contendo, 2% de extrato de levedura e 20% de sacarose. Dois métodos de extraçäo foram avaliados a partir do peso seco do micélico do fungo Aspergillus flavus e da quantidade de aflatoxina B produzida. Os extratos proteícos das três variedades testadas estimularam o crescimento micelial do fungo. Obteve-se uma maior concentraçäo proteíca nos extratos previamente tratados com acetona. Contudo, os dois métodos se mostraram igualmente viáveis como instrumento na diferenciaçäo varietal do milho, caracterizando a suscetibilidade de "Pipoca Americana" com altos níveis de aflatoxina produzida. "Centralmex N" e "Centralmex O2" apresentaram uma menor produçäo de aflatoxina que o controle. Os resultados sugerem que a composiçäo proteíca do milho pode apresentar elementos tanto eliciadores como inibidores à produçäo de aflatoxina B