First report of Sw-5 resistance-breaking strain of tomato spotted wilt orthotospovirus infecting tomato in Texas.

Tomato spotted wilt orthotospovirus (TSWV) is one of the most devastating plant viruses causing crop disease epidemics of global economic significance. A single dominant resistant gene 'Sw-5' offering a broad-spectrum resistance to multiple orthotospoviruses was introduced in tomato cultivars. However, multiple resistance-breaking strains of TSWV were reported worldwide (Ciuffo 2005; Zaccardelli et al. 2008; Batuman et al. 2017; di Rienzo et al. 2018). Symptoms suggestive of orthotospoviral infection including stunting, bronzing, and inward rolling of leaves, and concentric necrotic spots on leaves, petioles, and fruits were observed in two TSWV-resistant tomato cultivars ('BL163' and 'HT 2') planted in a tomato variety trial in Bushland, TX in 2022. Leaf tissues from 45 resistant tomato plants (symptomatic or asymptomatic) from both resistant cultivars were tested using a TaqMan probe-based qPCR assay targeting a 200bp region in nucleoprotein (N) of the TSWV (Gautam et al. 2022). While 25 of those samples tested positive for TSWV, only ten expressed characteristic disease symptoms described above. The possibility of mixed infection in those samples with other endemic viruses in the region viz., alfalfa mosaic virus, groundnut ringspot orthotospovirus, tobacco mosaic virus, tomato chlorotic spot orthotospovirus, tomato mosaic virus, tomato necrotic streak virus, tomato ringspot virus, and tomato torrado virus was discounted through RT-PCR analysis (Kumar et al. 2011; Verbeek et al. 2012; Bratsch et al. 2018). To test the RB phenotype of the observed putative TSWV-RB strains, three-week-old tomato plants from eight commercially available TSWV resistant cultivars and one non-resistant cultivar (n=10 each) were mechanically inoculated with leaf tissues collected from a single symptomatic plant from one of the field-grown resistant cultivars. The experiment was replicated twice. Hypersensitive response was observed on all inoculated leaves of resistant plants one week post inoculation. Furthermore, all eight resistant cultivars started expressing local and systemic TSW symptoms 12 to 16 days post inoculation (dpi), while non-resistant cultivar started expressing symptoms at 9 dpi. TSW incidence across all resistant cultivars was 30-70%, while in susceptible cultivar it was 90%. Symptoms exhibited by all resistant cultivars resembled those of symptoms observed in field collected plants. The expression of Sw-5 gene in all eight resistant cultivars and the lack thereof in a susceptible cultivar was confirmed using Sw-5b specific primers and using Actin as a housekeeping gene in qRT-PCR (Islam et al. 2022). The RB strains in Sw-5 resistant tomato in California (Batuman et al. 2017) had the C118Y mutation in the TSWV NSm protein, consistent with the original reporting of C118Y or T120N RB mutations in 11 TSWV isolates from Spain (NCBI accession # HM015517 & HM015518) (Lopez et al. 2011). The nucleotide and amino acid sequence analysis of NSm gene from Bushland RB isolates from four resistant cultivars (NCBI accessions # OP810513-14 [field], OQ247901-05 [mechanically inoculated]) shared 98.9 and 99.4% homology with the Californian NSm sequences of TSWV RB tomato isolate (KX898453 and ASO67371), respectively. While the Nsm C118Y or T120N RB mutations were absent in all Bushland TSWV RB isolates, they had six additional unique point mutations across the NSm (I163V, P227Q, V290I, N293S, V294I, K296Q), which could potentially be responsible for resistance breaking. Despite the lack of C118Y or T120N RB mutations, Bushland isolates were capable of disrupting Sw-5-mediated TSWV resistance in all eight commercial resistant tomato cultivars. This study suggests a new or a different class of fundamental mechanisms are likely to be responsible for resistance breaking in Sw-5b resistant tomatoes. The new RB strain/s of TSWV therefore pose a substantial threat to tomato production in TX and other tomato-growing regions of the US.

Autophagy-associated immune dysregulation and hyperplasia in a patient with compound heterozygous mutations in ATG9A.

ABBREVIATIONS: BCL2: BCL2 apoptosis regulator; BCL10: BCL10 immune signaling adaptor; CARD11: caspase recruitment domain family member 11; CBM: CARD11-BCL10-MALT1; CR2: complement C3d receptor 2; EBNA: Epstein Barr nuclear antigen; EBV: Epstein-Barr virus; FCGR3A; Fc gamma receptor IIIa; GLILD: granulomatous-lymphocytic interstitial lung disease; HV: healthy volunteer; IKBKB/IKB kinase: inhibitor of nuclear factor kappa B kinase subunit beta; IL2RA: interleukin 2 receptor subunit alpha; MALT1: MALT1 paracaspase; MS4A1: membrane spanning 4-domain A1; MTOR: mechanistic target of rapamycin kinase; MYC: MYC proto-oncogene, bHLH: transcription factor; NCAM1: neural cell adhesion molecule 1; NFKB: nuclear factor kappa B; NIAID: National Institute of Allergy and Infectious Diseases; NK: natural killer; PTPRC: protein tyrosine phosphatase receptor type C; SELL: selectin L; PBMCs: peripheral blood mononuclear cells; TR: T cell receptor; Tregs: regulatory T cells; WT: wild-type.

Separation of nordihydrocapsiate from capsiate and major capsaicinoid analogues using ultra high performance liquid chromatography.

Accurate, rapid quantitation of the capsaicinoid and capsinoid compounds produced by peppers (Capsicum spp.) is essential to assess quality. Here, we developed a rapid ultra-high performance liquid chromatography method for the simultaneous separation of five major capsaicinoids and three major capsinoids from peppers. Optimal chromatographic separation was achieved using a phenyl-hexyl stationary phase with a mobile phase of acidified water and methanol with a flow rate of 0.5 ml/min at a column temperature of 55 °C over 5 min. The method was validated by testing linearity, precision, robustness, and limits of detection and quantification. The developed method was successfully employed to profile capsaicinoids and capsinoids from different pepper cultivars. Out of the 10 pepper cultivars analysed, all three major capsinoids were detected in two cultivars. To the best of our knowledge, this is the first report of successful separation of nordihydrocapsiate from capsiate and quantification of nordihydrocapsiate.

Assessment and Classification of Volatile Profiles in Melon Breeding Lines Using Headspace Solid-Phase Microextraction Coupled with Gas Chromatography-Mass Spectrometry.

Cucumis melo L is one of the most commercial and economical crops in the world with several health beneficial compounds as such carotenoids, amino acids, vitamin A and C, minerals, and dietary fiber. Evaluation of the volatile organic compounds (VOCs) in different melon (Cucumis melo L.) breeding lines provides useful information for improving fruit flavor, aroma, and antimicrobial levels. In this study, the VOCs in 28 melon breeding lines harvested in 2019 were identified and characterized using head space solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). This identified 113 VOCs with significant differences in composition and contents of among the breeding lines, including 15 esters, 27 aldehydes, 35 alcohols, 14 ketones, 4 acids, 10 hydrocarbons, 5 sulfurs, and 3 other compounds. The highest average contents of all the VOCs were found in BL-30 (13,973.07 µg/kg FW) and the lowest were in BL-22 (3947.13 µg/kg FW). BL-9 had high levels of carotenoid-derived VOCs. The compounds with the highest contents were benzaldehyde, geranylacetone, and ß-ionone. Quality parameters such as color and sugar contents of melons were also measured. All the melon color readings were within the typical acceptable range. BL-22 and BL-14 had the highest and lowest sugar contents, respectively. Principal component analysis (PCA) produced diverse clusters of breeding lines based on flavor and aroma. BL-4, BL-7, BL-12, BL-20, and BL-30 were thus selected as important breeding lines based on their organoleptic, antimicrobial, and health-beneficial properties.

Frequent asymptomatic infection with tobacco ringspot virus on melon fruit.

Melon is one of the most popular fruits worldwide and has been bred into various cultivars. RNA-sequencing using healthy melon fruit was performed to determine differences in gene expression among cultivars. Unexpected RNA-seq results revealed that viruses asymptomatically infected fruits at a high frequency (16 of 21 fruits examined were infected) and that viral transcripts highly accumulated in comparison with host transcripts (15 %-75 % of total reads). Their nucleotide sequences and phylogenetic analyses indicated that more than 10 novel isolates of tobacco ringspot virus (TRSV) were found in melon fruits. Asymptomatic infection with TRSV on melon fruits was confirmed by both immunoblot and RT-PCR analyses. Numerous isolates of TRSV generated and maintained in melon fields, and this is likely due to their asymptomatic infections. This TRSV melon isolate infected Nicotiana benthamiana plants with stunting and yellowing symptoms. This is the first report of frequent and asymptomatic infection of TRSV in consumable melon fruits.

Effect of production system and inhibitory potential of aroma volatiles on polyphenol oxidase and peroxidase activity in tomatoes.

BACKGROUND: Polyphenol oxidase (PPO) and peroxidase (POD) are key enzymes associated with shelf life and defense responses. Thus, the activity of PPO and POD enzymes is usually assessed to check the quality of food samples and to understand the physiological responses of plants to different stresses. However, the outcomes of PPO and POD activity assessment studies are highly dependent on assay conditions. Hence, in this study, we initially optimized PPO and POD extraction and high-throughput 96-well plates-based enzymatic activity assessment methods to evaluate the inhibitory potential of tomato volatile compounds. Later, we explored the effects of net-house and open-field growing on the PPO and POD activity in tomato fruits of eight cultivars. RESULTS: We found 150 mM of catechol and pH 7.0 were the optimal conditions for the maximum activity for the PPO assay. Conversely, 24 mM guaiacol with 12 mM H2 O2 and pH 6.0 was the best condition for the POD assay. Thermal inactivation studies confirmed that tomato POD is more resistant to heat than PPO. We found that the production systems had a considerable genotype-specific impact on tomato PPO and POD activity. Moreover, amongst the volatiles that were studied, ß-damascenone and d-limonene showed 50% PPO inhibition at 40 and 80 mM, respectively. CONCLUSION: The results of this study can be used to improve the shelf-life of fresh tomato fruit and its products. The findings also underscore the significance of PPO and POD enzymes as physiological trait markers in the tomato crop and fruit quality improvement programs. © 2020 Society of Chemical Industry.

AKAP79/150 recruits the transcription factor NFAT to regulate signaling to the nucleus by neuronal L-type Ca2+ channels.

In neurons, regulation of activity-dependent transcription by the nuclear factor of activated T-cells (NFAT) depends upon Ca2+ influx through voltage-gated L-type calcium channels (LTCC) and NFAT translocation to the nucleus following its dephosphorylation by the Ca2+-dependent phosphatase calcineurin (CaN). CaN is recruited to the channel by A-kinase anchoring protein (AKAP) 79/150, which binds to the LTCC C-terminus via a modified leucine-zipper (LZ) interaction. Here we sought to gain new insights into how LTCCs and signaling to NFAT are regulated by this LZ interaction. RNA interference-mediated knockdown of endogenous AKAP150 and replacement with human AKAP79 lacking its C-terminal LZ domain resulted in loss of depolarization-stimulated NFAT signaling in rat hippocampal neurons. However, the LZ mutation had little impact on the AKAP-LTCC interaction or LTCC function, as measured by Förster resonance energy transfer, Ca2+ imaging, and electrophysiological recordings. AKAP79 and NFAT coimmunoprecipitated when coexpressed in heterologous cells, and the LZ mutation disrupted this association. Critically, measurements of NFAT mobility in neurons employing fluorescence recovery after photobleaching and fluorescence correlation spectroscopy provided further evidence for an AKAP79 LZ interaction with NFAT. These findings suggest that the AKAP79/150 LZ motif functions to recruit NFAT to the LTCC signaling complex to promote its activation by AKAP-anchored calcineurin.

Evaluation of bitter melon (Momordica charantia) cultivars grown in Texas and levels of various phytonutrients.

BACKGROUND: In the USA, Momordica charantia is relatively unknown and is usually found in specialty markets. In the present study, cultivation of five bitter melon cultivars grown under field conditions in College Station (TX, USA), was evaluated. Additionally, ascorbic acid, amino acids and phenolic compounds were quantified from various cultivars grown in different years. RESULTS: The yield of the first year of evaluation was comparable to other bitter melon growing regions, ranging from 9371.5 kg ha-1 for the Japanese Spindle cultivar to 20 839.1 kg ha-1 for the Hong Kong Green cultivar. Multivariate analysis suggests a strong correlation between yield and growth degree days, water use efficiency and organic matter, as well as an inverse correlation with the amount or precipitation during the growing season. The highest levels of total ascorbic acid were shown in the Japanese Spindle cultivar (162.97 mg 100 g-1 fresh fruit), whereas the lowest levels were expressed in the Hong Kong Green cultivar (42.69 mg 100 g-1 fresh fruit). The highest levels of total phenolics were consistently found the Indian White cultivar, in the range 10.6-12.5 mg g-1 catechin equivalents. Seven phenolics and organic acids were identified and quantified by liquid chromatography-mass spectrometry and high-performance liquid chromatography, respectively. Additionally, the highest levels of total amino acids were found in the Large Top cultivar. CONCLUSION: The current 3-year field study demonstrates that it is feasible to grow bitter melon commercially in Texas with proper climatic and agronomic conditions. Bitter melon is a rich source for ascorbic acid, amino acids and phenolic compounds, which makes it a valuable food source with respect to improving human health. © 2018 Society of Chemical Industry.

AKAP150 Palmitoylation Regulates Synaptic Incorporation of Ca2+-Permeable AMPA Receptors to Control LTP.

Ca2+-permeable AMPA-type glutamate receptors (CP-AMPARs) containing GluA1 but lacking GluA2 subunits contribute to multiple forms of synaptic plasticity, including long-term potentiation (LTP), but mechanisms regulating CP-AMPARs are poorly understood. A-kinase anchoring protein (AKAP) 150 scaffolds kinases and phosphatases to regulate GluA1 phosphorylation and trafficking, and trafficking of AKAP150 itself is modulated by palmitoylation on two Cys residues. Here, we developed a palmitoylation-deficient knockin mouse to show that AKAP150 palmitoylation regulates CP-AMPAR incorporation at hippocampal synapses. Using biochemical, super-resolution imaging, and electrophysiological approaches, we found that palmitoylation promotes AKAP150 localization to recycling endosomes and the postsynaptic density (PSD) to limit CP-AMPAR basal synaptic incorporation. In addition, we found that AKAP150 palmitoylation is required for LTP induced by weaker stimulation that recruits CP-AMPARs to synapses but not stronger stimulation that recruits GluA2-containing AMPARs. Thus, AKAP150 palmitoylation controls its subcellular localization to maintain proper basal and activity-dependent regulation of synaptic AMPAR subunit composition.

The fidelity of stochastic single-molecule super-resolution reconstructions critically depends upon robust background estimation.

The quality of super resolution images obtained by stochastic single-molecule microscopy critically depends on image analysis algorithms. We find that the choice of background estimator is often the most important determinant of reconstruction quality. A variety of techniques have found use, but many have a very narrow range of applicability depending upon the characteristics of the raw data. Importantly, we observe that when using otherwise accurate algorithms, unaccounted background components can give rise to biases on scales defeating the purpose of super-resolution microscopy. We find that a temporal median filter in particular provides a simple yet effective solution to the problem of background estimation, which we demonstrate over a range of imaging modalities and different reconstruction methods.

Influence of extraction solvents on antioxidant activity and the content of bioactive compounds in non-pungent peppers.

Bioactive compounds in foods have been shown to maintain human health. However, the relative amounts of bioactive compounds and the variation in the amounts are still poorly understood. In this study, the efficacy of different extraction solvents (hexane, ethyl acetate, acetone, methanol, and a methanol:water mixture), as well as the levels of certain bioactive compounds in non-pungent pepper cultivars (TMH, TMJ, PA137, and B58) were investigated using high-performance liquid chromatography (HPLC). Antioxidant activities were determined using 2,2,-diphenyl-1-picrylhydrazyl (DPPH), reducing power, and deoxyribose degradation. Hexane extracts had the highest level of carotenoids (47.2-628.8 µg/g), and methanol extracts contained maximum flavonoids (24.9-152.2 µg/g) in four different cultivars. Higher DPPH scavenging activity was found in the hexane extracts from TMH, TMJ, PA137, and B58 (IC50 value: 0.67, 0.74, 0.55, and 0.48 µg/ml, respectively), whereas the reducing power was high in ethyl acetate and acetone extracts. Inhibition of deoxyribose degradation was highest in methanolic extracts from TMH, TMJ, PA137, and B58 (51.2, 49.5, 52.6, and 47.4 %, respectively). These data demonstrate that solvent chemical properties such as polarity can differentially impact the efficiency with which different bioactive compounds are recovered from foods, and this could lead to differences in estimated biological activity such as antioxidant capacity.