Effector loss drives adaptation of Pseudomonas syringae pv. actinidiae biovar 3 to Actinidia arguta.

A pandemic isolate of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has devastated kiwifruit orchards growing cultivars of Actinidia chinensis. In contrast, A. arguta (kiwiberry) is not a host of Psa3. Resistance is mediated via effector-triggered immunity, as demonstrated by induction of the hypersensitive response in infected A. arguta leaves, observed by microscopy and quantified by ion-leakage assays. Isolates of Psa3 that cause disease in A. arguta have been isolated and analyzed, revealing a 51 kb deletion in the exchangeable effector locus (EEL). This natural EEL-mutant isolate and strains with synthetic knockouts of the EEL were more virulent in A. arguta plantlets than wild-type Psa3. Screening of a complete library of Psa3 effector knockout strains identified increased growth in planta for knockouts of four effectors-AvrRpm1a, HopF1c, HopZ5a, and the EEL effector HopAW1a -suggesting a resistance response in A. arguta. Hypersensitive response (HR) assays indicate that three of these effectors trigger a host species-specific HR. A Psa3 strain with all four effectors knocked out escaped host recognition, but a cumulative increase in bacterial pathogenicity and virulence was not observed. These avirulence effectors can be used in turn to identify the first cognate resistance genes in Actinidia for breeding durable resistance into future kiwifruit cultivars.

Real-Time PCR and Droplet Digital PCR Are Accurate and Reliable Methods To Quantify Pseudomonas syringae pv. actinidiae Biovar 3 in Kiwifruit Infected Plantlets.

Pseudomonas syringae pv. actinidiae is the etiological agent of kiwifruit canker disease, causing severe economic losses in kiwifruit production areas around the world. Rapid diagnosis, understanding of bacterial virulence, and rate of infection in kiwifruit cultivars are important in applying effective measures of disease control. P. syringae pv. actinidiae load in kiwifruit is currently determined by a labor-intense colony counting method with no high-throughput and specific quantification method being validated. In this work, we used three alternative P. syringae pv. actinidiae quantification methods in two infected kiwifruit cultivars: start of growth time, quantitative PCR (qPCR), and droplet digital PCR (ddPCR). Method performance in each case was compared with the colony counting method. Methods were validated using calibration curves obtained with serial dilutions of P. syringae pv. actinidiae biovar 3 (Psa3) inoculum and standard growth curves obtained from kiwifruit samples infected with Psa3 inoculum. All three alternative methods showed high correlation (r > 0.85) with the colony counting method. qPCR and ddPCR were very specific, sensitive (5 × 102 CFU/cm2), highly correlated to each other (r = 0.955), and flexible, allowing for sample storage. The inclusion of a kiwifruit biomass marker increased the methods' accuracy. The qPCR method was efficient and allowed for high-throughput processing, and the ddPCR method showed highly accurate results but was more expensive and time consuming. While not ideal for high-throughput processing, ddPCR was useful in developing accurate standard curves for the qPCR method. The combination of the two methods is high-throughput, specific for Psa3 quantification, and useful for research studies (e.g., disease phenotyping and host-pathogen interactions).

Carbon starvation reduces carbohydrate and anthocyanin accumulation in red-fleshed fruit via trehalose 6-phosphate and MYB27.

Kiwifruit (Actinidia spp.) is a recently domesticated fruit crop with several novel-coloured cultivars being developed. Achieving uniform fruit flesh pigmentation in red genotypes is challenging. To investigate the cause of colour variation between fruits, we focused on a red-fleshed Actinidia chinensis var. chinensis genotype. It was hypothesized that carbohydrate supply could be responsible for this variation. Early in fruit development, we imposed high or low (carbon starvation) carbohydrate supplies treatments; carbohydrate import or redistribution was controlled by applying a girdle at the shoot base. Carbon starvation affected fruit development as well as anthocyanin and carbohydrate metabolite concentrations, including the signalling molecule trehalose 6-phosphate. RNA-Seq analysis showed down-regulation of both gene-encoding enzymes in the anthocyanin and carbohydrate biosynthetic pathways. The catalytic trehalose 6-phosphate synthase gene TPS1.1a was down-regulated, whereas putative regulatory TPS7 and TPS11 were strongly up-regulated. Unexpectedly, under carbon starvation MYB10, the anthocyanin pathway regulatory activator was slightly up-regulated, whereas MYB27 was also up-regulated and acts as a repressor. To link these two metabolic pathways, we propose a model where trehalose 6-phosphate and the active repressor MYB27 are involved in sensing the carbon starvation status. This signals the plant to save resources and reduce the production of anthocyanin in fruits.

Multiple quantitative trait loci contribute to resistance to bacterial canker incited by Pseudomonas syringae pv. actinidiae in kiwifruit (Actinidia chinensis).

Pseudomonas syringae pv. actinidiae (Psa) biovar 3, a virulent, canker-inducing pathogen is an economic threat to the kiwifruit (Actinidia spp.) industry worldwide. The commercially grown diploid (2×) A. chinensis var. chinensis is more susceptible to Psa than tetraploid and hexaploid kiwifruit. However information on the genetic loci modulating Psa resistance in kiwifruit is not available. Here we report mapping of quantitative trait loci (QTLs) regulating resistance to Psa in a diploid kiwifruit population, derived from a cross between an elite Psa-susceptible 'Hort16A' and a resistant male breeding parent P1. Using high-density genetic maps and intensive phenotyping, we identified a single QTL for Psa resistance on Linkage Group (LG) 27 of 'Hort16A' revealing 16-19% phenotypic variance and candidate alleles for susceptibility and resistance at this loci. In addition, six minor QTLs were identified in P1 on distinct LGs, exerting 4-9% variance. Resistance in the F1 population is improved by additive effects from 'Hort16A' and P1 QTLs providing evidence that divergent genetic pathways interact to combat the virulent Psa strain. Two different bioassays further identified new QTLs for tissue-specific responses to Psa. The genetic marker at LG27 QTL was further verified for association with Psa resistance in diploid Actinidia chinensis populations. Transcriptome analysis of Psa-resistant and susceptible genotypes in field revealed hallmarks of basal defense and provided candidate RNA-biomarkers for screening for Psa resistance in greenhouse conditions.

A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants.

BACKGROUND: Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) 'Hongyang' draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models. RESULTS: A second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within 'Hongyang' The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned 'Hort16A' cDNAs and comparing with the predicted protein models for Red5 and both the original 'Hongyang' assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised 'Hongyang' annotation, respectively, compared with 90.9% to the Red5 models. CONCLUSIONS: Our study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and correction of gene models enabling improvement of computational prediction. This utility was especially relevant for certain types of gene families such as the EXPANSIN like genes. Finally, this high quality gene set will supply the kiwifruit and general plant community with a new tool for genomics and other comparative analysis.

A novel hairpin library-based approach to identify NBS-LRR genes required for effector-triggered hypersensitive response in Nicotiana benthamiana.

BACKGROUND: PTI and ETI are the two major defence mechanisms in plants. ETI is triggered by the detection of pathogen effectors, or their activity, in the plant cell and most of the time involves internal receptors known as resistance (R) genes. An increasing number of R genes responsible for recognition of specific effectors have been characterised over the years; however, methods to identify R genes are often challenging and cannot always be translated to crop plants. RESULTS: We present a novel method to identify R genes responsible for the recognition of specific effectors that trigger a hypersensitive response (HR) in Nicotiana benthamiana. This method is based on the genome-wide identification of most of the potential R genes of N. benthamiana and a systematic silencing of these potential R genes in a simple transient expression assay. A hairpin-RNAi library was constructed covering 345 R gene candidates of N. benthamiana. This library was then validated using several previously described R genes. Our approach indeed confirmed that Prf, NRC2a/b and NRC3 are required for the HR that is mediated in N. benthamiana by Pto/avrPto (prf, NRC2a/b and NRC3) and by Cf4/avr4 (NRC2a/b and NRC3). We also confirmed that NRG1, in association with N, is required for the Tobacco Mosaic Virus (TMV)-mediated HR in N. benthamiana. CONCLUSION: We present a novel approach combining bioinformatics, multiple-gene silencing and transient expression assay screening to rapidly identify one-to-one relationships between pathogen effectors and host R genes in N. benthamiana. This approach allowed the identification of previously described R genes responsible for detection of avirulence determinants from Pseudomonas, Cladosporium and TMV, demonstrating that the method could be applied to any effectors/proteins originating from a broad range of plant pathogens that trigger an HR in N. benthamiana. Moreover, with the increasing availability of genome sequences from model and crop plants and pathogens, this approach could be implemented in other plants, accelerating the process of identification and characterization of novel resistance genes.

In the Solanaceae, a hierarchy of bHLHs confer distinct target specificity to the anthocyanin regulatory complex.

The anthocyanin biosynthetic pathway is regulated by a transcription factor complex consisting of an R2R3 MYB, a bHLH, and a WD40. Although R2R3 MYBs belonging to the anthocyanin-activating class have been identified in many plants, and their role well elucidated, the subgroups of bHLH implicated in anthocyanin regulation seem to be more complex. It is not clear whether these potential bHLH partners are biologically interchangeable with redundant functions, or even if heterodimers are involved. In this study, AcMYB110, an R2R3 MYB isolated from kiwifruit (Actinidia sp.) showing a strong activation of the anthocyanin pathway in tobacco (Nicotiana tabacum) was used to examine the function of interacting endogenous bHLH partners. Constitutive expression of AcMYB110 in tobacco leaves revealed different roles for two bHLHs, NtAN1 and NtJAF13. A hierarchical mechanism is shown to control the regulation of transcription factors and consequently of the anthocyanin biosynthetic pathway. Here, a model is proposed for the regulation of the anthocyanin pathway in Solanaceous plants in which AN1 is directly involved in the activation of the biosynthetic genes, whereas JAF13 is involved in the regulation of AN1 transcription.

A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots.

Plants require sophisticated regulatory mechanisms to ensure the degree of anthocyanin pigmentation is appropriate to myriad developmental and environmental signals. Central to this process are the activity of MYB-bHLH-WD repeat (MBW) complexes that regulate the transcription of anthocyanin genes. In this study, the gene regulatory network that regulates anthocyanin synthesis in petunia (Petunia hybrida) has been characterized. Genetic and molecular evidence show that the R2R3-MYB, MYB27, is an anthocyanin repressor that functions as part of the MBW complex and represses transcription through its C-terminal EAR motif. MYB27 targets both the anthocyanin pathway genes and basic-helix-loop-helix (bHLH) ANTHOCYANIN1 (AN1), itself an essential component of the MBW activation complex for pigmentation. Other features of the regulatory network identified include inhibition of AN1 activity by the competitive R3-MYB repressor MYBx and the activation of AN1, MYB27, and MYBx by the MBW activation complex, providing for both reinforcement and feedback regulation. We also demonstrate the intercellular movement of the WDR protein (AN11) and R3-repressor (MYBx), which may facilitate anthocyanin pigment pattern formation. The fundamental features of this regulatory network in the Asterid model of petunia are similar to those in the Rosid model of Arabidopsis thaliana and are thus likely to be widespread in the Eudicots.

Endogenous cytokinin in developing kiwifruit is implicated in maintaining fruit flesh chlorophyll levels.

BACKGROUND AND AIMS: Green kiwifruit (Actinidia deliciosa) retain high concentrations of chlorophyll in the fruit flesh, whereas in gold-fleshed kiwifruit (A. chinensis) chlorophyll is degraded to colourless catabolites during fruit development, leaving yellow carotenoids visible. The plant hormone group the cytokinins has been implicated in the delay of senescence, and so the aim of this work was to investigate the link between cytokinin levels in ripening fruit and chlorophyll de-greening. METHODS: The expression of genes related to cytokinin metabolism and signal transduction and the concentration of cytokinin metabolites were measured. The regulation of gene expression was assayed using transient activation of the promoter of STAY-GREEN2 (SGR2) by cytokinin response regulators. KEY RESULTS: While the total amount of cytokinin increased in fruit of both species during maturation and ripening, a high level of expression of two cytokinin biosynthetic gene family members, adenylate isopentenyltransferases, was only detected in green kiwifruit fruit during ripening. Additionally, high levels of O-glucosylated cytokinins were detected only in green kiwifruit, as was the expression of the gene for zeatin O-glucosyltransferase, the enzyme responsible for glucosylating cytokinin into a storage form. Season to season variation in gene expression was seen, and some de-greening of the green kiwifruit fruit occurred in the second season, suggesting environmental effects on the chlorophyll degradation pathway. Two cytokinin-related response regulators, RRA17 and RRB120, showed activity against the promoter of kiwifruit SGR2. CONCLUSIONS: The results show that in kiwifruit, levels of cytokinin increase markedly during fruit ripening, and that cytokinin metabolism is differentially regulated in the fruit of the green and gold species. However, the causal factor(s) associated with the maintenance or loss of chlorophyll in kiwifruit during ripening remains obscure.

An R2R3 MYB transcription factor determines red petal colour in an Actinidia (kiwifruit) hybrid population.

BACKGROUND: Red colour in kiwifruit results from the presence of anthocyanin pigments. Their expression, however, is complex, and varies among genotypes, species, tissues and environments. An understanding of the biosynthesis, physiology and genetics of the anthocyanins involved, and the control of their expression in different tissues, is required. A complex, the MBW complex, consisting of R2R3-MYB and bHLH transcription factors together with a WD-repeat protein, activates anthocyanin 3-O-galactosyltransferase (F3GT1) to produce anthocyanins. We examined the expression and genetic control of anthocyanins in flowers of Actinidia hybrid families segregating for red and white petal colour. RESULTS: Four inter-related backcross families between Actinidia chinensis Planch. var. chinensis and Actinidia eriantha Benth. were identified that segregated 1:1 for red or white petal colour. Flower pigments consisted of five known anthocyanins (two delphinidin-based and three cyanidin-based) and three unknowns. Intensity and hue differed in red petals from pale pink to deep magenta, and while intensity of colour increased with total concentration of anthocyanin, no association was found between any particular anthocyanin data and hue. Real time qPCR demonstrated that an R2R3 MYB, MYB110a, was expressed at significant levels in red-petalled progeny, but not in individuals with white petals.A microsatellite marker was developed that identified alleles that segregated with red petal colour, but not with ovary, stamen filament, or fruit flesh colour in these families. The marker mapped to chromosome 10 in Actinidia.The white petal phenotype was complemented by syringing Agrobacterium tumefaciens carrying Actinidia 35S::MYB110a into the petal tissue. Red pigments developed in white petals both with, and without, co-transformation with Actinidia bHLH partners. MYB110a was shown to directly activate Actinidia F3GT1 in transient assays. CONCLUSIONS: The transcription factor, MYB110a, regulates anthocyanin production in petals in this hybrid population, but not in other flower tissues or mature fruit. The identification of delphinidin-based anthocyanins in these flowers provides candidates for colour enhancement in novel fruits.

The control of chlorophyll levels in maturing kiwifruit.

Chlorophyll is present in many plant organs, including immature fruit where it is usually degraded during ripening. Mature green kiwifruit (Actinidia deliciosa) are an exception, with high concentrations of chlorophyll remaining in the fruit flesh. In gold-fleshed kiwifruit (A. chinensis), chlorophyll is degraded to colourless catabolites upon fruit ripening, leaving yellow carotenoids visible. We have identified candidate genes for the control of chlorophyll degradation in kiwifruit and examined the transcript levels of these genes in maturing kiwifruit using quantitative real-time PCR. Results indicate that the biosynthesis and degradation, or turnover, of chlorophyll is transcriptionally regulated in green- and gold-fleshed kiwifruit. Both species of kiwifruit were found to have two homologues of the stay-green gene (SGR), a small protein that is postulated to aid in the dismantling of the light-harvesting complex, allowing free chlorophyll to enter the degradation pathway. However, with the exception of very mature green fruit, where degreening was observed, SGR2 was more highly expressed in gold fruit, indicating a potential regulatory step of chlorophyll degradation. When the SGR genes were over-expressed in tobacco leaves, degreening was observed. Our results show that chlorophyll degradation is differentially regulated in kiwifruit, and suggest that gold kiwifruit transcribe more degradation genes, leading to earlier and more sustained chlorophyll degradation in this fruit than in green kiwifruit.

Identification and characterisation of F3GT1 and F3GGT1, two glycosyltransferases responsible for anthocyanin biosynthesis in red-fleshed kiwifruit (Actinidia chinensis).

Much of the diversity of anthocyanins is due to the action of glycosyltransferases, which add sugar moieties to anthocyanidins. We identified two glycosyltransferases, F3GT1 and F3GGT1, from red-fleshed kiwifruit (Actinidia chinensis) that perform sequential glycosylation steps. Red-fleshed genotypes of kiwifruit accumulate anthocyanins mainly in the form of cyanidin 3-O-xylo-galactoside. Genes in the anthocyanin and flavonoid biosynthetic pathway were identified and shown to be expressed in fruit tissue. However, only the expression of the glycosyltransferase F3GT1 was correlated with anthocyanin accumulation in red tissues. Recombinant enzyme assays in vitro and in vivo RNA interference (RNAi) demonstrated the role of F3GT1 in the production of cyanidin 3-O-galactoside. F3GGT1 was shown to further glycosylate the sugar moiety of the anthocyanins. This second glycosylation can affect the solubility and stability of the pigments and modify their colour. We show that recombinant F3GGT1 can catalyse the addition of UDP-xylose to cyanidin 3-galactoside. While F3GGT1 is responsible for the end-product of the pathway, F3GT1 is likely to be the key enzyme regulating the accumulation of anthocyanin in red-fleshed kiwifruit varieties.

Characterization and quantification of anthocyanins in red kiwifruit ( Actinidia spp.).

Red-fleshed fruit occur in a small number of distantly related taxa in different sections of the genus Actinidia (kiwifruit). We describe and identify the anthocyanin profile of fruit of several Actinidia species. Differences in the relative amounts of cyanidin- and delphinidin-based anthocyanins determine whether the fruit appear red or purple. Cyanidin derivatives have been found in all Actinidia species that contain anthocyanins, whereas delphinidin derivatives are limited to two taxa: A. melanandra and A. arguta var. purpurea . The fruit of these not only contain a wider range of anthocyanins, but they also have greater concentrations. Anthocyanins of most Actinidia species are usually conjugated with either xylosyl-galactose or galactose, whereas A. deliciosa anthocyanins are conjugated with glucose and galactose.

The kiwifruit lycopene beta-cyclase plays a significant role in carotenoid accumulation in fruit.

The composition of carotenoids, along with anthocyanins and chlorophyll, accounts for the distinctive range of colour found in the Actinidia (kiwifruit) species. Lutein and beta-carotene are the most abundant carotenoids found during fruit development, with beta-carotene concentration increasing rapidly during fruit maturation and ripening. In addition, the accumulation of beta-carotene and lutein is influenced by the temperature at which harvested fruit are stored. Expression analysis of carotenoid biosynthetic genes among different genotypes and fruit developmental stages identified Actinidia lycopene beta-cyclase (LCY-beta) as the gene whose expression pattern appeared to be associated with both total carotenoid and beta-carotene accumulation. Phytoene desaturase (PDS) expression was the least variable among the different genotypes, while zeta carotene desaturase (ZDS), beta-carotene hydroxylase (CRH-beta), and epsilon carotene hydroxylase (CRH-epsilon) showed some variation in gene expression. The LCY-beta gene was functionally tested in bacteria and shown to convert lycopene and delta-carotene to beta-carotene and alpha-carotene respectively. This indicates that the accumulation of beta-carotene, the major carotenoid in these kiwifruit species, appears to be controlled by the level of expression of LCY-beta gene.

Isolation and structural identification of the anthocyanin components of red kiwifruit.

The anthocyanins responsible for the red color of red kiwifruit were extracted in acidified ethanol and isolated by solid phase extraction (SPE) followed by preparative HPLC. Five anthocyanins were obtained and subsequently identified as delphinidin 3-[2-(xylosyl)galactoside], delphinidin 3-galactoside, cyanidin 3-[2-(xylosyl)galactoside], cyanidin 3-galactoside, and cyanidin 3-glucoside by a combination of LC-MS/MS, GC-MS, and 2D NMR. Delphinidin 3-[2-(xylosyl)galactoside] and delphinidin 3-galactoside have not previously been reported in the genus Actinidia.

Analysis of expressed sequence tags from Actinidia: applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening.

BACKGROUND: Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). RESULTS: The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. CONCLUSION: This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Pigments in the fruit of red-fleshed kiwifruit (Actinidia chinensis and Actinidia deliciosa).

Kiwifruit cultivars (Actinidia chinensis and A. deliciosa) generally have fruit with yellow or green flesh when ripe. A small number of genotypes also have red pigments, usually restricted to the inner pericarp but varying in intensity and in distribution within the fruit. Carotenoids, chorophylls, and anthocyanins were extracted from the fruit pericarp of such red-fleshed kiwifruit selections. Pigments were analyzed by HPLC and identified by comparison with authentic standards and by liquid chromatography-mass spectroscopy to obtain a tentative identification of the major anthocyanins in red-fleshed kiwifruit. The yellow and green colors of the outer fruit pericarp are due to different concentrations and proportions of carotenoids and chlorophylls. The red color found mainly in the inner pericarp is due to anthocyanins. In the A. chinensis genotypes tested the major anthocyanin was cyanidin 3-O-xylo(1-2)-galactoside, with smaller amounts of cyanidin 3-O-galactoside. In the A. deliciosa genotypes analyzed, cyanidin 3-O-xylo(1-2)-galactoside was not detected; instead, the major anthocyanins identified were cyanidin 3-O-galactoside and cyanidin 3-O-glucoside. However, the two species did not differ consistently in anthocyanin composition.