Calmodulin-like protein CML15 interacts with PP2C46/65 to regulate papaya fruit ripening via integrating calcium, ABA and ethylene signals.

It is well known that calcium, ethylene and abscisic acid (ABA) can regulate fruit ripening, however, their interaction in the regulation of fruit ripening has not yet been fully clarified. The present study found that the expression of the papaya calcium sensor CpCML15 was strongly linked to fruit ripening. CpCML15 could bind Ca2+ and served as a true calcium sensor. CpCML15 interacted with CpPP2C46 and CpPP2C65, the candidate components of the ABA signalling pathways. CpPP2C46/65 expression was also related to fruit ripening and regulated by ethylene. CpCML15 was located in the nucleus and CpPP2C46/65 were located in both the nucleus and membrane. The interaction between CpCML15 and CpPP2C46/65 was calcium dependent and further repressed the activity of CpPP2C46/65 in vitro. The transient overexpression of CpCML15 and CpPP2C46/65 in papaya promoted fruit ripening and gene expression related to ripening. The reduced expression of CpCML15 and CpPP2C46/65 by virus-induced gene silencing delayed fruit colouring and softening and repressed the expression of genes related to ethylene signalling and softening. Moreover, ectopic overexpression of CpCML15 in tomato fruit also promoted fruit softening and ripening by increasing ethylene production and enhancing gene expression related to ripening. Additionally, CpPP2C46 interacted with CpABI5, and CpPP2C65 interacted with CpERF003-like, two transcriptional factors in ABA and ethylene signalling pathways that are closely related to fruit ripening. Taken together, our results showed that CpCML15 and CpPP2Cs positively regulated fruit ripening, and their interaction integrated the cross-talk of calcium, ABA and ethylene signals in fruit ripening through the CpCML15-CpPP2Cs-CpABI5/CpERF003-like pathway.

Mutualistic interaction of native Serratia marcescens UENF-22GI with Trichoderma longibrachiatum UENF-F476 boosting seedling growth of tomato and papaya.

A plethora of bacteria-fungal interactions occur on the extended fungal hyphae network in soil. The mycosphere of saprophytic fungi can serve as a bacterial niche boosting their survival, dispersion, and activity. Such ecological concepts can be converted to bioproducts for sustainable agriculture. Accordingly, we tested the hypothesis that the well-characterised beneficial bacterium Serratia marcescens UENF-22GI can enhance plant growth-promoting properties when combined with Trichoderma longibrachiatum UENF-F476. The cultural and cell interactions demonstrated S. marcescens and T. longibrachiatum mutual compatibility. Bacteria cells were able to attach, forming aggregates to biofilms and migrating through the fungal hyphae network. Long-distance bacterial migration through growing hyphae was confirmed using a two-compartment Petri dishes assay. Fungal inoculation increased the bacteria survival rates into the vermicompost substrate over the experimental time. Also, in vitro indolic compound, phosphorus, and zinc solubilisation bacteria activities increased in the presence of the fungus. In line with the ecophysiological bacteria fitness, the bacterium-fungal combination boosted tomato and papaya plantlet growth when applied into the plant substrate under nursery conditions. Mutualistic interaction between mycosphere-colonizing bacterium S. marcescens UENF-22GI and the saprotrophic fungi T. longibrachiatum UENF-F467 increased the ecological fitness of the bacteria alongside with beneficial potential for plant growth. A proper combination and delivery of mutual compatible beneficial bacteria-fungal represent an open avenue for microbial-based products for the biological enrichment of plant substrates in agricultural systems.

Metabolomic and Transcriptomic Profiling Provide Novel Insights into Fruit Ripening and Ripening Disorder Caused by 1-MCP Treatments in Papaya.

Treatment with 1-methylcyclopropylene (1-MCP) is an effective technique to preserve fruits, but inappropriate treatment with 1-MCP causes a ripening disorder (rubbery texture) in papaya fruit. In this study, a combined metabolomic and transcriptomic analysis was conducted to reveal the possible mechanism of the ripening disorder caused by unsuitable 1-MCP in papaya. A total of 203 differential accumulated metabolites (DAMs) were identified in the metabolome analysis. Only 24 DAMs were identified in the control (CK) vs. the 1-MCP 2 h group, and they were primarily flavonoids. Ninety and 89 DAMs were identified in the CK vs. 1-MCP 16 h and 1-MCP 2 h vs. 1-MCP 16 h groups, respectively, indicating that long-term 1-MCP treatment severely altered the metabolites during fruit ripening. 1-MCP 16 h treatment severely reduced the number of metabolites, which primarily consisted of flavonoids, lipids, phenolic acids, alkaloids, and organic acids. An integrated analysis of RNA-Seq and metabolomics showed that various energy metabolites for the tricarboxylic acid cycle were reduced by long-term treatment with 1-MCP, and the glycolic acid cycle was the most significantly affected, as well as the phenylpropane pathway. These results provide valuable information for fruit quality control and new insight into the ripening disorder caused by unsuitable treatment with 1-MCP in papaya.

Discovery of SNPs and InDels in papaya genotypes and its potential for marker assisted selection of fruit quality traits.

Papaya is a tropical and climacteric fruit that is recognized for its nutritional benefits and medicinal applications. Its fruits ripen quickly and show a drastic fruit softening, leading to great post-harvest losses. To overcome this scenario, breeding programs of papaya must invest in exploring the available genetic variation to continue developing superior cultivars with improved fruit quality traits. The objective of this study was to perform a whole-genome genotyping (WGG) of papaya, predict the effects of the identified variants, and develop a list of ripening-related genes (RRGs) with linked variants. The Formosa elite lines of papaya Sekati and JS-12 were submitted to WGG with an Illumina Miseq platform. The effects of variants were predicted using the snpEff program. A total of 28,451 SNPs having Ts/Tv (Transition/Transversion) ratio of 2.45 and 1,982 small insertions/deletions (InDels) were identified. Most variant effects were predicted in non-coding regions, with only 2,104 and 138 effects placed in exons and splice site regions, respectively. A total of 106 RRGs were found to be associated with 460 variants, which may be converted into PCR markers to facilitate genetic mapping and diversity studies and to apply marker-assisted selection (MAS) for specific traits in papaya breeding programs.

Stage-specific protein regulation during somatic embryo development of Carica papaya L. 'Golden'.

Somatic embryogenesis is an important biotechnological technique for large-scale propagation of elite genotypes. Identifying stage-specific compounds associated with somatic embryo development can help elucidate the ontogenesis of Carica papaya L. somatic embryos and improve tissue culture protocols. To identify the stage-specific proteins that are present during the differentiation of C. papaya somatic embryos, proteomic analyses of embryos at the globular, heart, torpedo and cotyledonary developmental stages were performed. Mass spectrometry data have been deposited in the ProteomeXchange with the dataset identifier PXD021107. Comparative proteomic analyses revealed a total of 801 proteins, with 392 classified as differentially accumulated proteins in at least one of the developmental stages. The globular-staged presented a higher number of unique proteins (16), and 7 were isoforms of 60S ribosomal proteins, suggesting high translational activity at the beginning of somatic embryogenesis. Proteins related to mitochondrial metabolism accumulated to a high degree at the early developmental stages and then decreased with increasing development, and they contributed to cell homeostasis in early somatic embryos. A progressive increase in the accumulation of vicilin, late embryogenesis abundant proteins and chloroplastic proteins that lead to somatic embryo maturation was also observed. The differential accumulation of acetylornithine deacetylase and S-adenosylmethionine synthase 2 proteins was correlated with increases in putrescine and spermidine contents, which suggests that both polyamines should be tested to determine whether they increase the conversion rates of globular- to cotyledonary-staged somatic embryos. Taken together, the results showed that somatic embryo development in C. papaya is regulated by the differential accumulation of proteins, with ribosomal and mitochondrial proteins more abundant during the early somatic embryo stages and seed maturation proteins more abundant during the late stages.

Correlation among PME activity, viscoelastic, and structural parameters for Carica papaya edible tissue along ripening.

Papaya fruit, widely consumed around the world, is mechanically and structurally affected by several enzymatic processes during ripening, where pectin methylesterase plays a key role. Hence, the aim of this work was to evaluate possible correlations among physicochemical changes, mechanical parameters, viscoelastic behavior, and enzyme activity of pectin methylesterase to provide information about the softening phenomenon by applying the Maxwell and Peleg models. Mechanical parameters were estimated by texture profile analysis, enzyme activity by Michaelis-Menten parameters, and viscoelastic behavior by relaxation test responses fitted to these models. The Maxwell model described properly mechanical changes during ripening, displaying a better adjustment (R2 > 0.97) than the Peleg model (0.80 < R2 < 0.84). Pearson correlation analysis (P ≤ 0.01) indicated an inversely proportional relation among firmness, total soluble solids, and the first elastic element of the Maxwell model. Besides, the PME Michaelis-Menten affinity constant showed a correlation between the first elastic element and the first viscoelastic element of the Maxwell model. Findings of this work pointed out that the first Maxwell elastic element could explain structural changes as papaya ripening advance, associated with pectin methylesterase activity, cell wall disruption, and cell assembling into the tissue. PRACTICAL APPLICATION: Mechanical and viscoelastic behavior of papaya fruit tissue were described by the Maxwell model associating both viscous and elastic elements to the softening process. The results provide background and practical knowledge to describe structural changes during the ripening process of papaya depending on its enzymatic activity. Outcomes could be further applied to understand changes in other fruits or food matrixes that soften during postharvest, storage, and food chain supply processes.

Two papaya MYB proteins function in fruit ripening by regulating some genes involved in cell-wall degradation and carotenoid biosynthesis.

BACKGROUND: MYB transcription factors (TFs) are common in plants and play important functions in growth and development, including fruit development and ripening. However, the role of MYB proteins in papaya ripening (fruit ripening and carotenoid biosynthesis) remains unclear. RESULTS: Two MYB genes were cloned from papaya pulp. They were named CpMYB1 (MYB44-like) and CpMYB2, and belong to the S22 subgroup of the R2R3-MYB family. Their expression levels decreased during fruit ripening. Subcellular localization analysis showed that both CpMYB1 and CpMYB2 were nuclear proteins, indicating that they might function in the nucleus. Moreover, CpMYB1 and CpMYB2 could bind to the promoters of cell-wall degradation genes (CpPME1, CpPME2, and CpPG5) and carotenoid biosynthesis genes (CpPDS2, CpPDS4, and CpCHY-b). Further research found that both CpMYB1 and CpMYB2 were transcriptional repressors, and they could suppress the activities of the promoters of CpPME1, CpPME2, CpPG5, CpPDS2, CpPDS4, and CpCHY-b. CONCLUSION: These results indicated that MYB TFs CpMYB1 and CpMYB2 might have a function in papaya fruit softening and carotenoid accumulation by regulating cell-wall degradation and carotenoid biosynthesis related genes, which provide a new view about the role of MYB TFs in fruit ripening. © 2020 Society of Chemical Industry.

CpARF2 and CpEIL1 interact to mediate auxin-ethylene interaction and regulate fruit ripening in papaya.

Papaya (Carica papaya L.) is a commercially important fruit crop. Various phytohormones, particularly ethylene and auxin, control papaya fruit ripening. However, little is known about the interaction between auxin and ethylene signaling during the fruit ripening process. In the present study, we determined that the interaction between the CpARF2 and CpEIL1 mediates the interaction between auxin and ethylene signaling to regulate fruit ripening in papaya. We identified the ethylene-induced auxin response factor CpARF2 and demonstrated that it is essential for fruit ripening in papaya. CpARF2 interacts with an important ethylene signal transcription factor CpEIL1, thus increasing the CpEIL1-mediated transcription of the fruit ripening-associated genes CpACS1, CpACO1, CpXTH12 and CpPE51. Moreover, CpEIL1 is ubiquitinated by CpEBF1 and is degraded through the 26S proteasome pathway. However, CpARF2 weakens the CpEBF1-CpEIL1 interaction and interferes with CpEBF1-mediated degradation of CpEIL1, promoting fruit ripening. Therefore, CpARF2 functions as an integrator in the auxin-ethylene interaction and regulates fruit ripening by stabilizing CpEIL1 protein and promoting the transcriptional activity of CpEIL1. To our knowledge, we have revealed a novel module of CpARF2/CpEIL1/CpEBF1 that fine-tune fruit ripening in papaya. Manipulating this mechanism could help growers tightly control papaya fruit ripening and prolong shelf life.

Roles of transcription factor SQUAMOSA promoter binding protein-like gene family in papaya (Carica papaya) development and ripening.

SQUAMOSA promoter binding protein-like (SPL) family plays vital regulatory roles in plant growth and development. The SPL family in climacteric fruit Carica papaya has not been reported. This study identified 14 papaya SPLs (CpSPL) from papaya genome and analyzed their sequence features, phylogeny, intron/exon structure, conserved motif, miR156-mediated posttranscriptional regulation, and expression patterns. 14 CpSPLs were clustered into 8 groups, and two distinct expression patterns were revealed for miR156-targeted and nontargeted CpSPLs in different tissues and fruit development stages. The expression changes of CpSPLs in ethephon and 1-MCP treated fruit during ripening suggested that the CpSPLs guided by CpmiR156 play crucial roles in ethylene signaling pathway. This study sheds light on the new function of SPL family in fruit development and ripening, providing insights on understanding evolutionary divergence of the members of SPL family among plant species.

Differential gene expression among three sex types reveals a MALE STERILITY 1 (CpMS1) for sex differentiation in papaya.

BACKGROUND: Carica papaya is a trioecious plant species with a genetic sex-determination system defined by sex chromosomes. Under unfavorable environmental conditions male and hermaphrodite exhibit sex-reversal. Previous genomic research revealed few candidate genes for sex differentiation in this species. Nevertheless, more analysis is still needed to identify the mechanism responsible for sex flower organ development in papaya. RESULTS: The aim of this study was to identify differentially expressed genes among male, female and hermaphrodite flowers in papaya during early (pre-meiosis) and later (post-meiosis) stages of flower development. RNA-seq was used to evaluate the expression of differentially expressed genes and RT-qPCR was used to verify the results. Putative functions of these genes were analyzed based on their homology with orthologs in other plant species and their expression patterns. We identified a Male Sterility 1 gene (CpMS1) highly up-regulated in male and hermaphrodite flower buds compared to female flower buds, which expresses in small male flower buds (3-8 mm), and that might be playing an important role in male flower organ development due to its homology to MS1 genes previously identified in other plants. This is the first study in which the sex-biased expression of genes related to tapetum development in the anther developmental pathway is being reported in papaya. Besides important transcription factors related to flower organ development and flowering time regulation, we identified differential expression of genes that are known to participate in ABA, ROS and auxin signaling pathways (ABA-8-hydroxylases, AIL5, UPBEAT 1, VAN3-binding protein). CONCLUSIONS: CpMS1 was expressed in papaya male and hermaphrodite flowers at early stages, suggesting that this gene might participate in male flower organ development processes, nevertheless, this gene cannot be considered a sex-determination gene. Due to its homology with other plant MS1 proteins and its expression pattern, we hypothesize that this gene participates in anther development processes, like tapetum and pollen development, downstream gender specification. Further gene functional characterization studies in papaya are required to confirm this hypothesis. The role of ABA and ROS signaling pathways in papaya flower development needs to be further explored as well.

Profit Analysis of Papaya Crops under Greenhouses as an Alternative to Traditional Intensive Horticulture in Southeast Spain.

The high-yield agricultural model in Almería is based on eight different crops. Having led fruit and vegetable exports in Spain for more than 50 years, a decrease in melon and watermelon growing areas in Almería caused a change in supply that affected the model's profit. Papaya cultivation could reactivate the profit of the agricultural model in Almería and also improve the available product range. The papaya crop needs greenhouse infrastructures high enough to contain the growth and size of the plants during a cycle crop, which is possible in most of the greenhouses of the Horticultural production model of Almería. The papaya harvests obtained in the region meet the quality requirements demanded by European markets. Furthermore, yields obtained are equal or higher than yields obtained by other producing countries. This crop improves profit compared with the profit obtained from the rotation of other horticultural crops that have been traditionally grown in the region.

Transcriptomic analysis reveals key factors in fruit ripening and rubbery texture caused by 1-MCP in papaya.

BACKGROUND: Ethylene promotes fruit ripening whereas 1-methylcyclopropene (1-MCP), a non-toxic antagonist of ethylene, delays fruit ripening via the inhibition of ethylene receptor. However, unsuitable 1-MCP treatment can cause fruit ripening disorders. RESULTS: In this study, we show that short-term 1-MCP treatment (400 nL•L- 1, 2 h) significantly delays papaya fruit ripening with normal ripening characteristics. However, long-term 1-MCP treatment (400 nL•L- 1, 16 h) causes a "rubbery" texture of fruit. The comparative transcriptome analysis showed that a total of 5529 genes were differently expressed during fruit ripening compared to freshly harvested fruits. Comprehensive functional enrichment analysis showed that the metabolic pathways of carbon metabolism, plant hormone signal transduction, biosynthesis of amino acids, and starch and sucrose metabolism are involved in fruit ripening. 1-MCP treatment significantly affected fruit transcript levels. A total of 3595 and 5998 differently expressed genes (DEGs) were identified between short-term 1-MCP, long-term 1-MCP treatment and the control, respectively. DEGs are mostly enriched in the similar pathway involved in fruit ripening. A large number of DEGs were also identified between long-term and short-term 1-MCP treatment, with most of the DEGs being enriched in carbon metabolism, starch and sucrose metabolism, plant hormone signal transduction, and biosynthesis of amino acids. The 1-MCP treatments accelerated the lignin accumulation and delayed cellulose degradation during fruit ripening. Considering the rubbery phenotype, we inferred that the cell wall metabolism and hormone signal pathways are closely related to papaya fruit ripening disorder. The RNA-Seq output was confirmed using RT-qPCR by 28 selected genes that were involved in cell wall metabolism and hormone signal pathways. CONCLUSIONS: These results showed that long-term 1-MCP treatment severely inhibited ethylene signaling and the cell wall metabolism pathways, which may result in the failure of cell wall degradation and fruit softening. Our results reveal multiple ripening-associated events during papaya fruit ripening and provide a foundation for understanding the molecular mechanisms underlying 1-MCP treatment on fruit ripening and the regulatory networks.

Histone Deacetylase CpHDA3 Is Functionally Associated with CpERF9 in Suppression of CpPME1/2 and CpPG5 Genes during Papaya Fruit Ripening.

Histone deacetylase (HDAC) performs important functions in plant growth and development, including fruit ripening. As a complex biological process, fruit ripening involves the histone acetylation modification of ripening-associated genes. Histone deacetylase genes (HDACs) have been well studied in Arabidopsis and rice, but the biological functions of HDACs in papaya are poorly understood. In the present work, three CpHDACs, belonging to the RPD3/HDA1 subfamily, were identified from papaya and named as CpHDA1, CpHDA2, and CpHDA3. CpHDA1 and CpHDA2 were induced by propylene, while CpHDA3 was propylene-repressed. Moreover, CpHDA3 protein could physically interact with CpERF9 and enhance the transcriptional repression activities of CpERF9 to downstream genes CpPME1, CpPME2 and CpPG5. Histone acetylation levels of CpPME1 and CpPG5 were increased during fruit ripening. Taken together, these results suggested that CpERF9 recruits CpHDA3 to form a histone deacetylase repressor complex to mediate pectin methylesterase and polygalacturonase genes expression during papaya fruit ripening and softening.

Comparative proteomic analysis provides novel insights into the regulation mechanism underlying papaya (Carica papaya L.) exocarp during fruit ripening process.

BACKGROUND: Papaya (Carica papaya L.) is a popular climacteric fruit, undergoing various physico-chemical changes during ripening. Although papaya is widely cultivated and consumed, few studies on the changes in metabolism during its ripening process at the proteasome level have been performed. Using a newly developed TMT-LCMS analysis, proteomes of papaya fruit at different ripening stages were investigated. RESULTS: In total, 3220 proteins were identified, of which 2818 proteins were quantified. The differential accumulated proteins (DAPs) exhibited various biological functions and diverse subcellular localizations. The KEGG enrichment analysis showed that various metabolic pathways were significantly altered, particularly in flavonoid and fatty acid metabolisms. The up-regulation of several flavonoid biosynthesis-related proteins may provide more raw materials for pigment biosynthesis, accelerating the color variation of papaya fruit. Variations in the fatty acid metabolism- and cell wall degradation-related proteins were investigated during the ripening process. Furthermore, the contents of several important fatty acids were determined, and increased unsaturated fatty acids may be associated with papaya fruit volatile formation. CONCLUSIONS: Our data may give an intrinsic explanation of the variations in metabolism during the ripening process of papaya fruit.

The Production and Quality of Different Varieties of Papaya Grown under Greenhouse in Short Cycle in Continental Europe.

In Europe, papaya consumption is growing due to its nutritional properties. The proximity of consumer markets to Southeast Spain allows fruits to be harvested at a more advanced stage of maturity compared to exporting countries from outside Europe, a timeline which improves the quality of the papaya. Experiments have been carried out to assess the adaptation of papaya to protected cropping systems (under greenhouse) in the region. In this paper, we showed the results obtained in an experiment with five varieties, taking the most cultivated variety as control, which was grafted on its own female rootstock, in addition to another four new varieties that were introduced. Transplanting was made with early sex-identified plants in the nursery. Cultivation was developed in a 446-day cycle, almost 15 months and fruits were always harvested from the soil, due to the height that the plant reached in that period. The best yield parameters and fruit characteristics were obtained from hermaphrodite Intenzza papaya grafted on female papaya rootstock, although there were also other varieties which gave results that made possible its cultivation under this production system.

Transcriptome-wide effect of DE-ETIOLATED1 (DET1) suppression in embryogenic callus of Carica papaya.

Papaya is one of the most nutritional fruits, rich in vitamins, carotenoids, flavonoids and other antioxidants. Previous studies showed phytonutrient improvement without affecting quality in tomato fruit and rapeseed through the suppression of DE-ETIOLATED-1 (DET1), a negative regulator in photomorphogenesis. This study is conducted to study the effects of DET1 gene suppression in papaya embryogenic callus. Immature zygotic embryos were transformed with constitutive expression of a hairpin DET1 construct (hpDET1). PCR screening of transformed calli and reverse transcription quantitative PCR (RT-qPCR) verified that DET1 gene downregulation in two of the positive transformants. High-throughput cDNA 3' ends sequencing on DET1-suppressed and control calli for transcriptomic analysis of global gene expression identified a total of 452 significant (FDR < 0.05) differentially expressed genes (DEGs) upon DET1 suppression. The 123 upregulated DEGs were mainly involved in phenylpropanoid biosynthesis and stress responses, compared to 329 downregulated DEGs involved in developmental processes, lipid metabolism, and response to various stimuli. This is the first study to demonstrate transcriptome-wide relationship between light-regulated pathway and secondary metabolite biosynthetic pathways in papaya. This further supports that the manipulation of regulatory gene involved in light-regulated pathway is possible for phytonutrient improvement of tropical fruit crops.

The effects of an osmoregulator, carbohydrates and polyol on maturation and germination of 'Golden THB' papaya somatic embryos.

This study evaluated the effect of osmoregulators and carbohydrates on the maturation and germination of somatic embryos of papaya 'Golden THB'. Cotyledon explants from papaya seedlings germinated in vitro on basal MS medium were cultured on somatic embryogenesis induction medium (IM) containing MS salts, myo-inositol, sucrose, agar and p-chlorophenoxyacetic acid. After 50 days, embryogenic calli were transferred onto maturation media (MM) for 45 additional days. For experiment 1, a MS-based medium supplemented with abscisic acid, activated charcoal and concentrations of PEG 6000 (0; 40; 50; 60 and 70 g L-1) was used, whereas for experiment 2 malt extract concentrations (0; 0.1; 0.2; 0.3 and 0.4 g L-1) were assessed. The normal cotyledonary somatic embryos produced in experiment 2 were transferred to the germination medium (GM). The GM consisted of full-strength MS medium, sucrose, agar and was supplemented with myo-inositol at varying concentrations (0; 0.275; 0.55 and 0.825 mM). The PEG concentrations tested impaired the maturation of 'Golden THB' papaya somatic embryos. The MM, supplemented with malt extract at 0.153 g L-1, promoted the greatest development of normal somatic embryos (18.28 SE calli-1), that is, two cotyledonary leaves produced 36.56 SE calli-1. The supplementation with 0.45 mM myo-inositol provided the highest germination percentage (47.42%) and conversion to emblings.

Profile of siRNAs derived from green fluorescent protein (GFP)-tagged Papaya leaf distortion mosaic virus in infected papaya plants.

We used green fluorescent protein (GFP)-tagged Papaya leaf distortion mosaic virus (PLDMV-GFP) to track PLDMV infection by fluorescence. The virus-derived small interfering RNAs (vsiRNAs) of PLDMV-GFP were characterized from papaya plants by next-generation sequencing. The foreign GFP gene inserted into the PLDMV genome was also processed as a viral gene into siRNAs by components involved in RNA silencing. The siRNAs derived from PLDMV-GFP accumulated preferentially as 21- and 22-nucleotide (nt) lengths, and most of the 5'-terminal ends were biased towards uridine (U) and adenosine (A). The single-nucleotide resolution map revealed that vsiRNAs were heterogeneously distributed throughout the PLDMV-GFP genome, and vsiRNAs derived from the sense strand were more abundant than those from the antisense strand. The hotspots were mainly distributed in the P1 and GFP coding region of the antisense strand. In addition, 979 papaya genes targeted by the most abundant 1000 PLDMV-GFP vsiRNAs were predicted and annotated using GO and KEGG classification. Results suggest that vsiRNAs play key roles in PLDMV-papaya interactions. These data on the characterization of PLDMV-GFP vsiRNAs will help to provide insight into the function of vsiRNAs and their host target regulation patterns.

Diversity of mucoralean fungi in soils of papaya (Carica papaya L.) producing regions in Mexico.

Mexico is the fifth largest producer of papaya worldwide and has recently reported problems with mucoralean fungi in this crop. These fungi are considered saprophytes in the soil and are ubiquitous in nature. In this work, they were isolated from soil in regions of intensive papaya cultivation in Mexico. Collections were made in the states of Colima, Oaxaca and Veracruz in Apr 2016. A total of 72 mucorales fungal isolates was obtained and morphologically characterized and then molecular characterization (28S ribosomal region) of 25 representative isolates was carried out. Phylogenetic analysis of the sequences confirmed the presence of the species Gilbertella persicaria, Rhizopus oryzae, Rhizopus stolonifer, Mucor circinelloides and Mucor hiemalis, which cause soft rot in papaya fruits, therefore, spores of these fungi found in the orchard soils can be considered as a constant source of contamination that affects healthy fruits. Additionally, Choanephora cucurbitarum, Mucor ellipsoideus, Rhizopus homothallicus, Rhizopus microsporus, Rhizopus schipperae, Lichteimia ramosa, Gongronella butleri, Cunninghamella bertholletiae and Cunninghamella blakesleeana were identified which are considered to have agricultural, biotechnological and medical importance.

Differential methylation and expression of HUA1 ortholog in three sex types of papaya.

Papaya is trioecious and an excellent system for studying sex determination and differentiation in plants. An ortholog of HUA1, CpHUA1, a gene controlling stamen and carpel development in Arabidopsis, was cloned and characterized in papaya. CpHUA1 consists of 12 exons with full genomic length of 19,313 bp in male AU9 and 19,312 bp in hermaphrodite SunUp, whereas the Arabidopsis HUA1 consists of 12 exons with full genomic length of 4300 bp. All the 324 SNPs between male and hermaphrodite varieties are in the 11th intron, which spans 8.5 kb. Quantitative RT-PCR revealed that CpHUA1 expression is highly elevated in carpels, suggesting that CpHUA1 may be involved in sex differentiation gene network. Southern blot analysis revealed a distinct restriction pattern in male AU9 compared to hermaphrodite Kapoho and SunUp, despite high DNA sequence identity and sharing of all but two EcoR I restriction sites in genomic CpHUA1 sequences of AU9 and SunUp. The methylation of cytosine at one restriction site in male but not in other two sex types may result in distinct restriction pattern of EcoR I in southern blot result. Bisulfite sequencing showed differential methylation of CpHUA1 among sex types, particularly the enrichment of sex-specific methylation in 9th and 11th intron. The methylation difference in cold stress induced male to hermaphrodite mutant mostly observed in the CHH context of CpHUA1, but no methylation difference detected in CHH context in other sex types, which may indicate the role of methylation in CHH context of CpHUA1 in temperature-related stress response and sex reversal.