Contrasting effector profiles between bacterial colonisers of kiwifruit reveal redundant roles converging on PTI-suppression and RIN4.

Testing effector knockout strains of the Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) for reduced in planta growth in their native kiwifruit host revealed a number of nonredundant effectors that contribute to Psa3 virulence. Conversely, complementation in the weak kiwifruit pathogen P. syringae pv. actinidifoliorum (Pfm) for increased growth identified redundant Psa3 effectors. Psa3 effectors hopAZ1a and HopS2b and the entire exchangeable effector locus (ΔEEL; 10 effectors) were significant contributors to bacterial colonisation of the host and were additive in their effects on virulence. Four of the EEL effectors (HopD1a, AvrB2b, HopAW1a and HopD2a) redundantly contribute to virulence through suppression of pattern-triggered immunity (PTI). Important Psa3 effectors include several redundantly required effectors early in the infection process (HopZ5a, HopH1a, AvrPto1b, AvrRpm1a and HopF1e). These largely target the plant immunity hub, RIN4. This comprehensive effector profiling revealed that Psa3 carries robust effector redundancy for a large portion of its effectors, covering a few functions critical to disease.

Actinidin diversity: discovery of common and selective substrates for actinidin isoforms and Actinidia cultivars.

The actinidin proteinase family has a striking sequence diversity; isoelectric points range from 3.9 to 9.3. The biological drive for this variation is thought to be actinidin's role as a defense-related protein. In this study we map mutations in the primary sequence onto the 3D structure of the protein and show that the region with the highest diversity is close to the substrate binding groove. Non-conservative substitutions in the active site determine substrate preference and therefore create problems for quantification of actinidin activity. Here we use a peptide substrate library to compare two actinidin isoforms, one from the kiwiberry cultivar 'Hortgem Tahi' (Actinidia arguta), and the other from the familiar kiwifruit cultivar 'Hayward' (Actinidia chinensis var. deliciosa). Among 360 octamer substrates we find one substrate (RVAAGSPI) with the useful property of being readily cleaved by all the functionally active actinidins in a set of A. arguta and A. chinensis var. deliciosa isoforms. In addition, we find that two substrates (LPPKSQPP & ILRDKDNT) have the ability to differentiate different isoforms from a single fruit. We compare actinidins from 'Hayward' and A. arguta for their ability to digest the allergenic gluten peptide (PFPQPQLPY) but find the peptide to be indigestible by all sources of actinidin. The ability to inactivate salivary amylase is shown to be a common trait in Actinidia cultivars due to proteolysis by actinidin and is particularly strong in 'Hortgem Tahi'. A mixture of 10% 'Hortgem Tahi' extract with 90% saliva inactivates 100% of amylase activity within 5 minutes. Conceivably, 'Hortgem Tahi' might lower the glycaemic response in a meal rich in cooked starch.

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.

Transcriptome Responses of Ripe Cherry Tomato Fruit Exposed to Chilling and Rewarming Identify Reversible and Irreversible Gene Expression Changes.

Tomato fruit stored below 12°C lose quality and can develop chilling injury upon subsequent transfer to a shelf temperature of 20°C. The more severe symptoms of altered fruit softening, uneven ripening and susceptibility to rots can cause postharvest losses. We compared the effects of exposure to mild (10°C) and severe chilling (4°C) on the fruit quality and transcriptome of 'Angelle', a cherry-type tomato, harvested at the red ripe stage. Storage at 4°C (but not at 10°C) for 27 days plus an additional 6 days at 20°C caused accelerated softening and the development of mealiness, both of which are commonly related to cell wall metabolism. Transcriptome analysis using RNA-Seq identified a range of transcripts encoding enzymes putatively involved in cell wall disassembly whose expression was strongly down-regulated at both 10 and 4°C, suggesting that accelerated softening at 4°C was due to factors unrelated to cell wall disassembly, such as reductions in turgor. In fruit exposed to severe chilling, the reduced transcript abundances of genes related to cell wall modification were predominantly irreversible and only partially restored upon rewarming of the fruit. Within 1 day of exposure to 4°C, large increases occurred in the expression of alternative oxidase, superoxide dismutase and several glutathione S-transferases, enzymes that protect cell contents from oxidative damage. Numerous heat shock proteins and chaperonins also showed large increases in expression, with genes showing peak transcript accumulation after different times of chilling exposure. These changes in transcript abundance were not induced at 10°C, and were reversible upon transfer of the fruit from 4 to 20°C. The data show that genes involved in cell wall modification and cellular protection have differential sensitivity to chilling temperatures, and exhibit different capacities for recovery upon rewarming of the fruit.

Rapid Methodologies for Assessing Pseudomonas syringae pv. actinidiae Colonization and Effector-Mediated Hypersensitive Response in Kiwifruit.

The infection of Pseudomonas syringae pv. actinidiae in kiwifruit is currently assessed by numerous methodologies, each with their own limitations. Most studies are based on either a laborious method of growth quantification of the pathogen or qualitative assessments by visual scoring following stem or cutting inoculation. Additionally, when assessing for resistance against specific pathogen effectors, confounding interactions between multiple genes in the pathogen can make mapping resistance phenotypes nearly impossible. Here, we present robust alternative methods to quantify pathogen load based on rapid bacterial DNA quantification by PCR, the use of Pseudomonas fluorescens, and a transient reporter eclipse assay for assessing resistance conferred by isolated bacterial avirulence genes. These assays compare well with bacterial plate counts to assess bacterial colonization as a result of plant resistance activation. The DNA-based quantification, when coupled with the P. fluorescens and reporter eclipse assays to independently identify bacterial avirulence genes, is rapid, highly reproducible, and scalable for high-throughput screens of multiple cultivars or genotypes. Application of these methodologies will allow rapid and high-throughput identification of resistant cultivars and the bacterial avirulence genes they recognize, facilitating resistance gene discovery for plant breeding programs.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Selenium Application During Radish (Raphanus sativus) Plant Development Alters Glucosinolate Metabolic Gene Expression and Results in the Production of 4-(methylseleno)but-3-enyl glucosinolate.

Selenium (Se) is an essential micronutrient for human health, entering the diet mainly through the consumption of plant material. Members of the Brassicaceae are Se-accumulators that can accumulate up to 1g Se kg-1 dry weight (DW) from the environment without apparent ill effect. The Brassicaceae also produce glucosinolates (GSLs), sulfur (S)-rich compounds that benefit human health. Radish (Raphanus sativus) has a unique GSL profile and is a Se-accumulating species that is part of the human diet as sprouts, greens and roots. In this report we describe the effects of Se-fertilisation on GSL production in radish during five stages of early development (from seed to mature salad greens) and on the transcript abundance of eight genes encoding enzymes involved in GSL metabolism. We tentatively identified (by tandem mass spectrometry) the selenium-containing glucosinolate, 4-(methylseleno)but-3-enyl glucosinolate, with the double bond geometry not resolved. Two related isothiocyanates were tentatively identified by Gas Chromatography-Mass Spectrometry as (E/Z?) isomers of 4-(methylseleno)but-3-enyl isothiocyanate. Se fertilisation of mature radish led to the presence of selenoglucosinolates in the seed. While GSL concentration generally reduced during radish development, GSL content was generally not affected by Se fertilisation, aside from the indole GSL, indol-3-ylmethyl glucosinolate, which increased on Se treatment, and the Se-GSLs, which significantly increased during development. The transcript abundance of genes involved in aliphatic GSL biosynthesis declined with Se treatment while that of genes involved in indole GSL biosynthesis tended to increase. APS kinase transcript abundance increased significantly in three of the four developmental stages following Se treatment. The remaining genes investigated were not significantly changed following Se treatment. We hypothesise that increased APS kinase expression in response to Se treatment is part of a general protection mechanism controlling the uptake of S and the production of S-containing compounds such as GSLs. The upregulation of genes encoding enzymes involved in indole GSL biosynthesis and a decrease in those involved in aliphatic GSL biosynthesis may be part of a similar mechanism protecting the plant's GSL complement whilst limiting the amount of Se-GSLs produced.

Selenium treatment differentially affects sulfur metabolism in high and low glucosinolate producing cultivars of broccoli (Brassica oleracea L.).

The effect of selenium (Se) application on the sulfur (S)-rich glucosinolate (GSL)-containing plant, broccoli (Brassica oleracea L. var. italica) was examined with a view to producing germplasm with increased Se and GSL content for human health, and to understanding the influence of Se on the regulation of GSL production. Two cultivars differing in GSL content were compared. Increased Se application resulted in an increase in Se uptake in planta, but no significant change in total S or total GSL content in either cultivar. Also no significant change was observed in the activity of ATP sulfurylase (ATPS, EC 2.7.7.4) or O-acetylserine(thiol) lyase (OASTL, EC 2.5.1.47) with increased Se application. However, in the first investigation of APS kinase (APSK, EC 2.7.1.25) expression in response to Se fertilisation, an increase in transcript abundance of one variant of APS kinase 1 (BoAPSK1A) was observed in both cultivars, and an increase in BoAPSK2 transcript abundance was observed in the low GSL producing cultivar. A mechanism by which increased APSK transcription may provide a means of controlling the content of S-containing compounds, including GSLs, following Se uptake is proposed.

Distribution of selenoglucosinolates and their metabolites in Brassica treated with sodium selenate.

In Brassica species, hydrolysis of (methylthio)glucosinolates produces sulfur-containing aglycons which have demonstrated anticancer benefits. Selenized Brassicaceae contain (methylseleno)glucosinolates and their selenium-containing aglycons. As a prelude to biological testing, broccoli, cauliflower, and forage rape plants were treated with sodium selenate and their tap roots, stems, leaves, and florets analyzed for selenoglucosinolates and their Se aglycons. Two new selenoglucosinolates were identified: glucoselenoraphanin in broccoli florets and glucoselenonasturtiin in forage rape roots. A new aglycon, selenoberteroin nitrile, was identified in forage rape. The major selenoglucosinolates were glucoselenoerucin in broccoli, glucoselenoiberverin in cauliflower, and glucoselenoerucin and glucoselenoberteroin in forage rape roots. In broccoli florets, the concentrations of selenglucosinolates exceeded those of their sulfur analogues. Fertilization with selenium slightly reduced (methylthio)glucosinolates and aglycons in the roots, but increased them in the florets, the leaves, and sometimes the stems. These discoveries provide a new avenue for investigating how consumption of Brassica vegetables and their organoselenides may promote human health.

Consumption of selenium-enriched broccoli increases cytokine production in human peripheral blood mononuclear cells stimulated ex vivo, a preliminary human intervention study.

SCOPE: Selenium (Se) is a micronutrient essential for human health, including immune function. Previous research indicates that Se supplementation may cause a shift from T helper (Th)1- to Th2-type immune responses. We aim to test the potential health promoting effects of Se-enriched broccoli. METHODS AND RESULTS: In a human trial, 18 participants consumed control broccoli daily for 3 days. After a 3-day wash-out period, the participants were provided with Se-enriched broccoli containing 200 µg of Se per serving for 3 days. Plasma and peripheral blood mononuclear cell (PBMC) samples were collected at the start and end of each broccoli feeding period for analysis of total Se and measurement of cytokine production from PBMC stimulated with antigens ex vivo. Plasma Se content remained consistent throughout the control broccoli feeding period and the baseline of the Se-enriched broccoli period (1.22 µmol/L) and then significantly increased following 3 days of Se-enriched broccoli feeding. Interleukin (IL-2, IL-4, IL-5, IL-13, and IL-22) production from PBMC significantly increased after 3 days of Se-enriched broccoli feeding compared with baseline. CONCLUSION: This study indicates that consumption of Se-enriched broccoli may increase immune responses toward a range of immune challenges.

Post-translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold-induced sweetening of potato tubers.

Cold-induced sweetening (CIS) is a serious post-harvest problem for potato tubers, which need to be stored cold to prevent sprouting and pathogenesis in order to maintain supply throughout the year. During storage at cold temperatures (below 10 °C), many cultivars accumulate free reducing sugars derived from a breakdown of starch to sucrose that is ultimately cleaved by acid invertase to produce glucose and fructose. When affected tubers are processed by frying or roasting, these reducing sugars react with free asparagine by the Maillard reaction, resulting in unacceptably dark-coloured and bitter-tasting product and generating the probable carcinogen acrylamide as a by-product. We have previously identified a vacuolar invertase inhibitor (INH2) whose expression correlates both with low acid invertase activity and with resistance to CIS. Here we show that, during cold storage, overexpression of the INH2 vacuolar invertase inhibitor gene in CIS-susceptible potato tubers reduced acid invertase activity, the accumulation of reducing sugars and the generation of acrylamide in subsequent fry tests. Conversely, suppression of vacuolar invertase inhibitor expression in a CIS-resistant line increased susceptibility to CIS. The results show that post-translational regulation of acid invertase by the vacuolar invertase inhibitor is an important component of resistance to CIS.

Selenoglucosinolates and their metabolites produced in Brassica spp. fertilised with sodium selenate.

Glucosinolates are sulphur-containing glycosides found in many Brassica spp. that are important because their aglycone hydrolysis products protect the plant from herbivores and exhibit anti-cancer properties in humans. Recently, synthetically produced selenium analogues have been shown to be more effective at suppressing cancers than their sulphur counterparts. Although selenium is incorporated into a number of Brassica amino acids and peptides, firm evidence has yet to be presented for the presence of selenium in the glucosinolates and their aglycones in planta. In this study broccoli and cauliflower florets, and roots of forage rape, all obtained from plants treated with sodium selenate, were analysed for the presence of organoselenides. GC-MS analysis of pentane/ether extracts identified six organoselenium compounds including selenium analogues of known myrosinase-derived Brassica volatiles: 4-(methylseleno)butanenitrile, 5-(methylseleno)pentanenitrile, 3-(methylseleno)propylisothiocyanate, 4-(methylseleno)butylisothiocyanate, and 5-(methylseleno)pentylisothiocyanate. LC-MS analysis of ethanolic extracts identified three selenoglucosinolates: 3-(methylseleno)propylglucosinolate (glucoselenoiberverin), 4-(methylseleno)butylglucosinolate (glucoselenoerucin), and 5-(methylseleno)pentylglucosinolate (glucoselenoberteroin). LC-MS/MS analysis was used to locate the position of the selenium atom in the selenoglucosinolate and indicates preferential incorporation of selenium via selenomethionine into the methylselenyl moiety rather than into the sulphate or ß-thioglucose groups. In forage rape, selenoglucosinolates and their aglycones (mainly isothiocyanates), occurred at concentrations up to 10% and 70%, respectively, of their sulphur analogues. In broccoli, concentrations of the selenoglucosinolates and their aglycones (mainly nitriles) were up to 60% and 1300%, respectively of their sulphur analogues. These findings indicate the potential for the incorporation of high levels of selenium into Brassica glucosinolates.

Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced sweetening resistance and includes spliced hybrid mRNA variants.

Cold storage of tubers of potato (Solanum tuberosum L.) compromises tuber quality in many cultivars by the accumulation of hexose sugars in a process called cold-induced sweetening. This is caused by the breakdown of starch to sucrose, which is cleaved to glucose and fructose by vacuolar acid invertase. During processing of affected tubers, the high temperatures involved in baking and frying cause the Maillard reaction between reducing sugars and free amino acids, resulting in the accumulation of acrylamide. cDNA clones with deduced proteins homologous to known invertase inhibitors were isolated and the two most abundant forms, termed INH1 and INH2, were shown to possess apoplastic and vacuolar localization, respectively. The INH2 gene showed developmentally regulated alternative splicing, so, in addition to the INH2α transcript encoding the full-length protein, two hybrid mRNAs (INH2ß*A and INH2ß*B) that encoded deduced vacuolar invertase inhibitors with divergent C-termini were detected, the result of mRNA splicing of an upstream region of INH2 to a downstream region of INH1. Hybrid RNAs are common in animals, where they may add to the diversity of the proteome, but are rarely described in plants. During cold storage, INH2α and the hybrid INH2ß mRNAs accumulated to higher abundance in cultivars resistant to cold-induced sweetening than in susceptible cultivars. Increased amounts of invertase inhibitor may contribute to the suppression of acid invertase activity and prevent cleavage of sucrose. Evidence for increased RNA splicing activity was detected in several resistant lines, a mechanism that in some circumstances may generate a range of proteins with additional functional capacity to aid adaptability.