Habituation to thaxtomin A increases resistance to common scab in 'Russet Burbank' potato.

Common scab is a potato disease characterized by the formation of scab-like lesions on the surface of potato tubers. The actinobacterium Streptomyces scabiei is the main causal agent of common scab. During infection, this bacterium synthesizes the phytotoxin thaxtomin A which is essential for the production of disease symptoms. While thaxtomin A can activate an atypical programmed cell death in plant cell suspensions, it is possible to gradually habituate plant cells to thaxtomin A to provide resistance to lethal phytotoxin concentrations. Potato 'Russet Burbank' calli were habituated to thaxtomin A to regenerate the somaclone RB9 that produced tubers more resistant to common scab than those obtained from the original cultivar. Compared to the Russet Burbank cultivar, somaclone RB9 generated up to 22% more marketable tubers with an infected tuber area below the 5% threshold. Enhanced resistance was maintained over at least two years of cultivation in the field. However, average size of tubers was significantly reduced in somaclone RB9 compared to the parent cultivar. Small RB9 tubers had a thicker phellem than Russet Burbank tubers, which may contribute to improving resistance to common scab. These results show that thaxtomin A-habituation in potato is efficient to produce somaclones with increased and durable resistance to common scab.

Molecular and Biochemical Examination of Spraing Disease in Potato Tuber in Response to Tobacco rattle virus Infection.

Field-grown tubers of potato were examined for infection by Tobacco rattle virus (TRV) and consequent production of corky ringspot or spraing symptoms. A microarray study identified genes that are differentially expressed in tuber tissue in response to TRV infection and to spraing production, suggesting that hypersensitive response (HR) pathways are activated in spraing-symptomatic tubers. This was confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) of a selected group of HR-related genes and by histochemical staining of excised tuber tissue with spraing symptoms. qRT-PCR of TRV in different regions of the same tuber slice showed that nonsymptomatic areas contained higher levels of virus relative to spraing-symptomatic areas. This suggests that spraing formation is associated with an active plant defense that reduces the level of virus in the infected tuber. Expression of two of the same plant defense genes was similarly upregulated in tubers that were infected with Potato mop-top virus, a virus that also induces spraing formation.

Toughing It Out--Disease-Resistant Potato Mutants Have Enhanced Tuber Skin Defenses.

Common scab, a globally important potato disease, is caused by infection of tubers with pathogenic Streptomyces spp. Previously, disease-resistant potato somaclones were obtained through cell selections against the pathogen's toxin, known to be essential for disease. Further testing revealed that these clones had broad-spectrum resistance to diverse tuber-invading pathogens, and that resistance was restricted to tuber tissues. The mechanism of enhanced disease resistance was not known. Tuber periderm tissues from disease-resistant clones and their susceptible parent were examined histologically following challenge with the pathogen and its purified toxin. Relative expression of genes associated with tuber suberin biosynthesis and innate defense pathways within these tissues were also examined. The disease-resistant somaclones reacted to both pathogen and toxin by producing more phellem cell layers in the tuber periderm, and accumulating greater suberin polyphenols in these tissues. Furthermore, they had greater expression of genes associated with suberin biosynthesis. In contrast, signaling genes associated with innate defense responses were not differentially expressed between resistant and susceptible clones. The resistance phenotype is due to induction of increased periderm cell layers and suberization of the tuber periderm preventing infection. The somaclones provide a valuable resource for further examination of suberization responses and its genetic control.

And the nasty ones lose in the end: foliar pathogenicity trades off with asexual transmission in the Irish famine pathogen Phytophthora infestans.

A trade-off between pathogenicity and transmission is often postulated to explain the persistence of pathogens over time. If demonstrated, it would help to predict the evolution of pathogenicity across cropping seasons, and to develop sustainable control strategies from this prediction. Unfortunately, experimental demonstration of such trade-offs in agricultural plant pathogens remains elusive. We measured asexual transmission of Phytophthora infestans isolates differing in pathogenicity in two sets of artificial infection experiments under controlled, semi-outdoor conditions. Higher foliar pathogenicity decreased mean daughter tuber weight, increased infection severity in daughter tubers, and increased stem mortality before emergence. The most pathogenic isolates thus suffer a double penalty for asexual transmission: a lower survival probability within small and severely infected tubers; and a lower infection probability of neighbouring healthy plants due to fewer infected stems produced by surviving tubers. Moderate tuber resistance favoured transmission of the least pathogenic isolates, while high levels of resistance almost abolished transmission of all isolates. These data demonstrate a trade-off between foliar pathogenicity and asexual transmission over seasons in P. infestans, which should stabilise pathogenicity over time in the potato late blight pathosystem and possibly favour clone replacement by less pathogenic lineages after demographic bottlenecks.

Pseudomonas fluorescens LBUM223 Increases Potato Yield and Reduces Common Scab Symptoms in the Field.

Common scab of potato, caused by pathogenic Streptomyces spp., is an important disease not efficiently controlled by current methods. We previously demonstrated that Pseudomonas fluorescens LBUM223 reduces common scab development under controlled conditions through phenazine-1-carboxylic (PCA) production, leading to reduced thaxtomin A production by the pathogen, a key pathogenicity and virulence factor. Here, we aimed at determining if LBUM223 is able to increase potato yield and control common scab under field conditions, while characterizing the biocontrol mechanisms involved. We investigated if a reduction in pathogen soil populations, activation of induced systemic resistance in potato, and/or changes in txtA gene expression, involved in thaxtomin A biosynthesis in pathogenic Streptomyces spp. were involved in common scab control by LBUM223. Common scab symptoms were significantly reduced and total tuber weight increased by 46% using biweekly applications of LBUM223. LBUM223 did not reduce pathogen soil populations, nor was potato systemic defense-related gene expression significantly altered between treatments. However, a significant down-regulation of txtA expression occurred in the geocaulosphere. This is the first demonstration that a Pseudomonas strain can directly alter the transcriptional activity of a key pathogenesis gene in a plant pathogen under field conditions, contributing to disease control.

Testing Taxonomic Predictivity of Foliar and Tuber Resistance to Phytophthora infestans in Wild Relatives of Potato.

Potato late blight, caused by the oomycete phytopathogen Phytophthora infestans, is a devastating disease found in potato-growing regions worldwide. Long-term management strategies to control late blight include the incorporation of host resistance to predominant strains. However, due to rapid genetic changes within pathogen populations, rapid and recurring identification and integration of novel host resistance traits is necessary. Wild relatives of potato offer a rich source of desirable traits, including late blight resistance, but screening methods can be time intensive. We tested the ability of taxonomy, ploidy, crossing group, breeding system, and geography to predict the presence of foliar and tuber late blight resistance in wild Solanum spp. Significant variation for resistance to both tuber and foliar late blight was found within and among species but there was no discernable predictive power based on taxonomic series, clade, ploidy, breeding system, elevation, or geographic location. We observed a moderate but significant correlation between tuber and foliar resistance within species. Although previously uncharacterized sources of both foliar and tuber resistance were identified, our study does not support an assumption that taxonomic or geographic data can be used to predict sources of late blight resistance in wild Solanum spp.

Potato Tuber Blight Resistance Phenotypes Correlate with RB Transgene Transcript Levels in an Age-Dependent Manner.

Plants have evolved strategies and mechanisms to detect and respond to pathogen attack. Different organs of the same plant may be subjected to different environments (e.g., aboveground versus belowground) and pathogens with different lifestyles. Accordingly, plants commonly need to tailor defense strategies in an organ-specific manner. Phytophthora infestans, causal agent of potato late blight disease, infects both aboveground foliage and belowground tubers. We examined the efficacy of transgene RB (known for conferring foliar late blight resistance) in defending against tuber late blight disease. Our results indicate that the presence of the transgene has a positive yet only marginally significant effect on tuber disease resistance on average. However, a significant association between transgene transcript levels and tuber resistance was established for specific transformed lines in an age-dependent manner, with higher transcript levels indicating enhanced tuber resistance. Thus, RB has potential to function in both foliage and tuber to impart late blight resistance. Our data suggest that organ-specific resistance might result directly from transcriptional regulation of the resistance gene itself.

New food from a potato somatic hybrid: nutritional equivalence and safety assessment by a feeding study on rats.

BACKGROUND: Potato tubers from the STBd somatic hybrid line that exhibited improved tolerance to salinity and resistance to fungal and PVY infections were characterised. They were compared for their chemical composition to the Spunta variety produced by conventional agronomic practices. This study aimed to compare nutritional value and safety by feeding rats with STBd or commercial tubers added to the standard diet (20/80 w/w). RESULTS: The analysis of soluble sugar, fat, fibre and ash content of tubers did not reveal any significant differences between the hybrid line and the control Spunta variety. Small differences were observed in dry matter, starch and protein content of hybrid potatoes in comparison to controls. However, all values were within normal ranges reported in the literature. The feeding study on rats showed that overall health, weight gain, food consumption, morphological aspects and weights of organs were comparable between rat groups fed the STBd hybrid and the Spunta variety. CONCLUSION: Taken together, 28 days of consumption of STBd hybrid potato did not exert any adverse effect on rats compared with commercial Spunta potato. The STBd potato line was therefore considered to be as safe for food utilisation as the commercial variety.

New method for the simultaneous analysis of types A and B trichothecenes by ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry in potato tubers inoculated with Fusarium sulphureum.

A reliable and sensitive method for rapid simultaneous determination of two type A (T-2 and diacetoxyscirpenol) and two type B (3-acetyldeoxynivalenol and Fusarenon X) trichothecenes was developed and successfully applied for detecting trichothecenes in potato tubers by ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry. The established method was further evaluated by determining the linearity (R ≥ 0.9995), recovery (113.28-77.97%), precision (relative standard deviation ≤ 5.89), and sensitivity (limit of detection, 0.002-0.005 µg/g; limit of quantitation, 0.005-0.015 µg/g). The method proved to be suitable for simultaneous determination of T-2, diacetoxyscirpenol, 3-acetyldeoxynivalenol, and Fusarenon X in potato tubers inoculated with Fusarium sulphureum . In addition, it was found that T-2, diacetoxyscirpenol, 3-acetyldeoxynivalenol, and Fusarenon X could be predominantly detected in the lesion, and the toxin could also be identified in tubers without any disease symptoms. The experimental results also indicated that the concentration of toxin in the susceptible cultivar (Longshu No. 3) was significantly higher than that in the resistant cultivar (Longshu No. 6).

Colonization patterns of an mCherry-tagged Pectobacterium carotovorum subsp. brasiliense strain in potato plants.

Pectobacterium carotovorum subsp. brasiliense is a newly identified member of the potato soft rot enterobacteriaceae. The pathogenesis of this pathogen is still poorly understood. In this study, an mCherry-P. carotovorum subsp. brasiliense-tagged strain was generated to study P. carotovorum subsp. brasiliense-potato plant interactions. Prior to use, the tagged strain was evaluated for in vitro growth, plasmid stability, and virulence on potato tubers and shown to be similar to the wild type. Four potato cultivars were evaluated for stem-based resistance against P. carotovorum subsp. brasiliense. Confocal laser-scanning microscopy and in vitro viable cell counts showed that P. carotovorum subsp. brasiliense is able to penetrate roots of a susceptible potato cultivar as early as 12 h postinoculation and migrate upward into aerial stem parts. Due to the phenotypic differences observed between tolerant and susceptible cultivars, a comparison of P. carotovorum subsp. brasiliense colonization patterns in these cultivars was undertaken. In the susceptible cultivar, P. carotovorum subsp. brasiliense cells colonized the xylem tissue, forming "biofilm-like" aggregates that led to occlusion of some of the vessels. In contrast, in the tolerant cultivar, P. carotovorum subsp. brasiliense appeared as free-swimming planktonic cells with no specific tissue localization. This suggests that there are resistance mechanisms in the tolerant cultivar that limit aggregation of P. carotovorum subsp. brasiliense in planta and, hence, the lack of symptom development in this cultivar.

Insights into organ-specific pathogen defense responses in plants: RNA-seq analysis of potato tuber-Phytophthora infestans interactions.

BACKGROUND: The late blight pathogen Phytophthora infestans can attack both potato foliage and tubers. Although interaction transcriptome dynamics between potato foliage and various pathogens have been reported, no transcriptome study has focused specifically upon how potato tubers respond to pathogen infection. When inoculated with P. infestans, tubers of nontransformed 'Russet Burbank' (WT) potato develop late blight disease while those of transgenic 'Russet Burbank' line SP2211 (+RB), which expresses the potato late blight resistance gene RB (Rpi-blb1), do not. We compared transcriptome responses to P. infestans inoculation in tubers of these two lines. RESULTS: We demonstrated the practicality of RNA-seq to study tetraploid potato and present the first RNA-seq study of potato tuber diseases. A total of 483 million paired end Illumina RNA-seq reads were generated, representing the transcription of around 30,000 potato genes. Differentially expressed genes, gene groups and ontology bins that exhibited differences between the WT and +RB lines were identified. P. infestans transcripts, including those of known effectors, were also identified. CONCLUSION: Faster and stronger activation of defense related genes, gene groups and ontology bins correlate with successful tuber resistance against P. infestans. Our results suggest that the hypersensitive response is likely a general form of resistance against the hemibiotrophic P. infestans-even in potato tubers, organs that develop below ground.

Development of somatic hybrids Solanum × michoacanum Bitter. (Rydb.) (+) S. tuberosum L. and autofused 4x S. × michoacanum plants as potential sources of late blight resistance for potato breeding.

KEY MESSAGE: Phytophthora infestans resistant somatic hybrids of S. × michoacanum (+) S. tuberosum and autofused 4 x S. × michoacanum were obtained. Our material is promising to introgress resistance from S. × michoacanum into cultivated potato background. Solanum × michoacanum (Bitter.) Rydb. (mch) is a wild diploid (2n = 2x = 24) potato species derived from spontaneous cross of S. bulbocastanum and S. pinnatisectum. This hybrid is a 1 EBN (endosperm balance number) species and can cross effectively only with other 1 EBN species. Plants of mch are resistant to Phytophthora infestans (Mont) de Bary. To introgress late blight resistance genes from mch into S. tuberosum (tbr), genepool somatic hybridization between mch and susceptible diploid potato clones (2n = 2x = 24) or potato cultivar Rywal (2n = 4x = 48) was performed. In total 18,775 calli were obtained from postfusion products from which 1,482 formed shoots. The Simple Sequence Repeat (SSR), Cleaved Amplified Polymorphic Sequences (CAPS) and Random Amplified Polymorphic DNA (RAPD) analyses confirmed hybrid nature of 228 plants and 116 autofused 4x mch. After evaluation of morphological features, flowering, pollen stainability, tuberization and ploidy level, 118 somatic hybrids and 116 autofused 4x mch were tested for late blight resistance using the detached leaf assay. After two seasons of testing three somatic hybrids and 109 4x mch were resistant. Resistant forms have adequate pollen stainability for use in crossing programme and are a promising material useful for introgression resistance from mch into the cultivated potato background.

Hydrophobic proteins secreted into the apoplast may contribute to resistance against Phytophthora infestans in potato.

During plant-pathogen interaction, oomycetes secrete effectors into the plant apoplast where they interact with host resistance proteins, which are accumulated after wounding or infection. Previous studies showed that the expression profile of pathogenesis related proteins is proportional to the resistance of different cultivars toward Phytophthora infestans infection. The aim of this work was to analyze the expression pattern of apoplastic hydrophobic proteins (AHPs), after 24 h of wounding or infection, in tubers from two potato cultivars with different resistance to P. infestans, Spunta (susceptible) and Innovator (resistant). Intercellular washing fluid (IWF) was extracted from tubers and chromatographed into a PepRPC™ HR5-5 column in FPLC eluted with a linear gradient of 75% acetonitrile. Then, AHPs were analyzed by SDS-PAGE and identified by MALDI-TOF-MS. Innovator cv. showed a higher basal AHP content compared to Spunta cv. In the latter, infection induced accumulation of patatins and protease inhibitors (PIs), whereas in Innovator cv. no changes in PIs accumulation were observed. In response to P. infestans infection, lipoxygenase, enolase, annexin p34 and glutarredoxin/cyclophilin were accumulated in both cultivars. These results suggest that the AHPs content may be related to the protection against the oomycete and with the degree of potato resistance to pathogens. Additionally, a considerable number of the proteins putatively identified lacked the signal peptide and, being SecretomeP positive, suggest unconventional protein secretion.

Quantitative resistance of potato to Pectobacterium atrosepticum and Phytophthora infestans: integrating PAMP-triggered response and pathogen growth.

While the mechanisms underlying quantitative resistance of plants to pathogens are still not fully elucidated, the Pathogen-Associated Molecular Patterns (PAMPs)-triggered response model suggests that such resistance depends on a dynamic interplay between the plant and the pathogen. In this model, the pathogens themselves or elicitors they produce would induce general defense pathways, which in turn limit pathogen growth and host colonisation. It therefore suggests that quantitative resistance is directly linked to a common set of general host defense mechanisms, but experimental evidence is still inconclusive. We tested the PAMP-triggered model using two pathogens (Pectobacterium atrosepticum and Phytophthora infestans) differing by their infectious processes and five potato cultivars spanning a range of resistance levels to each pathogen. Phenylalanine ammonia-lyase (PAL) activity, used as a defense marker, and accumulation of phenolics were measured in tuber slices challenged with lipopolysaccharides from P. atrosepticum or a concentrated culture filtrate from P. infestans. PAL activity increased following treatment with the filtrate but not with lipopolysaccharides, and varied among cultivars. It was positively related to tuber resistance to P. atrosepticum, but negatively related to tuber resistance to P. infestans. It was also positively related to the accumulation of total phenolics. Chlorogenic acid, the main phenolic accumulated, inhibited growth of both pathogens in vitro, showing that PAL induction caused active defense against each of them. Tuber slices in which PAL activity had been induced before inoculation showed increased resistance to P. atrosepticum, but not to P. infestans. Our results show that inducing a general defense mechanism does not necessarily result in quantitative resistance. As such, they invalidate the hypothesis that the PAMP-triggered model alone can explain quantitative resistance. We thus designed a more complex model integrating physiological host response and a key pathogen life history trait, pathogen growth, to explain the differences between the two pathosystems.

[Immunomodulating activity of chitosan derivatives with salicylic acid and its fragments].

A study of biological activity of the derivatives of the chitin-chitosan oligomer with salicylic acid and its fragments showed that chitosan salicylate actively protected potato tubers against Phytophthora infestans but sharply inhibited reparation of potato tissues. N-(2-Hydroxybenzyl)chitosan exhibited good protective properties but did not influence wound reparation. N-(2-Hydroxy-3-methoxybenzyl)-N-pyridox-chitosan, which contained the pyridoxal and 2-hydroxy-3-methoxy fragments, was the most efficient, stimulating both defense against late blight and wound reparation in potato tissues.

Green-tissue-specific, C(4)-PEPC-promoter-driven expression of Cry1Ab makes transgenic potato plants resistant to tuber moth (Phthorimaea operculella, Zeller).

An important strategy for obtaining a safer transgenic plant may be the use of a spatial- or tissue-specific promoter, instead of a constitutive one. In this study, we have used a light-inducible maize PEPC promoter to regulate the cry1Ab gene, aiming to produce transgenic potatoes that are resistant to potato tuber moth (PTM) (Phthorimaea operculella, Zeller). Out of 60 regenerated lines having normal phenotypes, 55 lines were PCR-positive for both the cry1Ab and nptII genes. Southern analysis on three selected putative transgenic lines revealed that they have only a single intact copy of the cry1Ab gene. An investigation of the Cry1Ab protein in the leaves and light-exposed (LE) tubers of the transgenic lines demonstrated the presence of the protein in the foliage and green tubers but not in the light-not exposed (LNE) tubers. A bioassay analysis of excised leaves of nine randomly selected lines showed that eight lines had 100% PTM larval mortality. Confirming results were obtained in six selected lines using the whole plant bioassay in the greenhouse. LE transgenic tubers also exhibited 100% larval mortality; however, the levels of damage to the LNE transgenic tubers were high and statistically the same as those incurred by the non-transgenic ones. Based on the results, we believe that this spatial expression of Cry1Ab using the light-inducible PEPC promoter can control PTM infestation in the field and significantly reduce pollution transmission to storage potatoes.

Immunomodulation of enzyme function in plants by single-domain antibody fragments.

Immunomodulation involves the use of antibodies to alter the function of molecules and is an emerging tool for manipulating both plant and animal systems. To realize the full potential of this technology, two major obstacles must be overcome. First, most antibodies do not function well intracellularly because critical disulfide bonds cannot form in the reducing environment of the cytoplasm or because of difficulties in targeting to subcellular organelles. Second, few antibodies bind to the active sites of enzymes and thus they generally do not neutralize enzyme function. Here we show that the unique properties of single-domain antibodies from camelids (camels and llamas) can circumvent both these obstacles. We demonstrate that these antibodies can be correctly targeted to subcellular organelles and inhibit enzyme function in plants more efficiently than antisense approaches. The use of these single-domain antibody fragments may greatly facilitate the successful immunomodulation of metabolic pathways in many organisms.