Structural and biochemical characterization of SmoPG1, an exo-polygalacturonase from Selaginella moellendorffii.

Polygalacturonases (PGs) can modulate chemistry and mechanical properties of the plant cell wall through the degradation of pectins, one of its major constituents. PGs are largely used in food, beverage, textile, and paper industries to increase processes' performances. To improve the use of PGs, knowledge of their biochemical, structural and functional features is of prime importance. Our study aims at characterizing SmoPG1, a polygalacturonase from Selaginella moellendorffii, that belongs to the lycophytes. Transcription data showed that SmoPG1 was mainly expressed in S. moellendorffii shoots while phylogenetic analyses suggested that SmoPG1 is an exo-PG, which was confirmed by the biochemical characterization following its expression in heterologous system. Indeed, LC-MS/MS oligoprofiling using various pectic substrates identified galacturonic acid (GalA) as the main hydrolysis product. We found that SmoPG1 was most active on polygalacturonic acid (PGA) at pH 5, and that its activity could be modulated by different cations (Ca2+, Cu2+, Fe2+, Mg2+, Mn2+, Na2+, Zn2+). In addition, SmoPG1 was inhibited by green tea catechins, including (-)-epigallocatechin-3-gallate (EGCG). Docking analyses and MD simulations showed in detail amino acids responsible for the SmoPG1-EGCG interaction. Considering its expression yield and activity, SmoPG1 appears as a prime candidate for the industrial production of GalA.

Biochemical characterization of a novel acid-active endopolygalacturonase for pectin depolymerization, pectic-oligomer production, and fruit juice clarification.

Endopolygalacturonases are crucial pectinases known for their efficient and sustainable pectin depolymerization activities. The present study identified a novel gene encoding endopolygalacturonase from an acidic mine tailing metagenome. The putative gene showed a maximum identity of 67.55 % with an uncharacterized peptide sequence from Flavobacterium fluvii. The gene was cloned and expressed in a heterologous host, E. coli. Biochemical characterization of the novel endopolygalacturonase enzyme variant (EPHM) showed maximum activity at 60 °C and at 5.0 pH, while retaining 50 % activity under the temperature and pH range of 20 °C to 70 °C for 6 h, and 3.0 to 10.0 for 3 h, respectively. The enzyme exhibited tolerance to different metal ions. EPHM was characterized for the depolymerization of methylated pectin into pectic oligosaccharides. Further, its utility was established for fruit juice clarification, as endorsed by high transmittance, significant viscosity reduction, and release of reducing sugars in the treated fruit juice samples.

Supplementation of citrus pectin with whole-cell pectinase PG5 on Pichia pastoris promotes recovery of colitis and enhances intestinal barrier function in DSS-treated mice.

A whole-cell biocatalyst was developed by genetically engineering pectinase PG5 onto the cell surface of Pichia pastoris using Gcw12 as the anchoring protein. Whole-cell PG5 eliminated the need for enzyme extraction and purification, while also exhibiting enhanced thermal stability, pH stability, and resistance to proteases in vitro compared to free PG5. Magnetic resonance mass spectrometry analysis revealed that whole-cell PG5 efficiently degraded citrus pectin, resulting in the production of a mixture of pectin oligosaccharides. The primary components of the mixture were trigalacturonic acid, followed by digalacturonic acid and tetragalacturonic acid. Supplementation of citrus pectin with whole-cell PG5 resulted in a more pronounced protective effect compared to free PG5 in alleviating colitis symptoms and promoting the integrity of the colonic epithelial barrier in a mouse model of dextran sulfate sodium-induced colitis. Hence, this study demonstrates the potential of utilizing whole-cell pectinase as an effective biocatalyst to promote intestinal homeostasis in vivo.

Application of Thermomyces lanuginosus polygalacturonase produced in Komagataella phaffii in biomass hydrolysis and textile bioscouring.

In this work, the polygalacturonase (TL-PG1) from the thermophilic fungus Thermomyces lanuginosus was heterologously produced for the first time in the yeast Komagataella phaffii. The TL-PG1 was successfully expressed under the control of the AOX1 promoter and sequentially purified by His-tag affinity. The purified recombinant pectinase exhibited an activity of 462.6 U/mL toward polygalacturonic acid under optimal conditions (pH 6 and 55 ˚C) with a 2.83 mg/mL and 0.063 µmol/minute for Km and Vmax, respectively. When used as supplementation for biomass hydrolysis, TL-PG1 demonstrated synergy with the enzymatic cocktail Ctec3 to depolymerize orange citrus pulp, releasing 1.43 mg/mL of reducing sugar. In addition, TL-PG1 exhibited efficiency in fabric bioscouring, showing potential usage in the textile industry. Applying a protein dosage of 7 mg/mL, the time for the fabric to absorb water was 19.77 seconds (ten times faster than the control). Adding the surfactant Triton to the treatment allowed the reduction of the enzyme dosage by 50% and the water absorption time to 6.38 seconds. Altogether, this work describes a new versatile polygalacturonase from T. lanuginosus with the potential to be employed in the hydrolysis of lignocellulosic biomass and bioscouring.

Enhancement of novel Endo-polygalacturonase expression in Rhodotorula mucilaginosa PY18: insights from mutagenesis and molecular docking.

Pectinase is a particular type of enzyme that can break down pectin compounds and is extensively utilised in the agricultural field. In this study, twenty yeast isolates were isolated and assayed for pectinase activity. Molecular identification by PCR amplification and sequencing of internal transcribed spacer (ITS) regions of isolate no. 18 had the highest pectinase activity of 46.35 U/mg, was identified as Rhodotorula mucilaginosa PY18, and was submitted under accession no. (OM275426) in NCBI. Rhodotorula mucilaginosa PY18 was further enhanced through sequential mutagenesis, resulting in a mutant designated as Rhodotorula mucilaginosa E54 with a specific activity of 114.2 U/mg. Using Response Surface Methodology (RSM), the best culture conditions for the pectinase-producing yeast mutant Rhodotorula mucilaginosa E54 were pH 5, 72-h incubation, 2.5% xylose, and 2.5% malt extract, with a pectinase-specific activity of 156.55 U/mg. Then, the obtained sequences of the endo-polygalacturonase PGI gene from Rhodotorula mucilaginosa PY18 and mutant Rhodotorula mucilaginosa E54 were isolated for the first time, sequenced, and submitted to NCBI accession numbers OQ283005 and OQ283006, respectively. The modelled 3D structure of the endo-PGI enzyme (485 residues) was validated using Ramachandran's plot, which showed 87.71, 85.56, and 91.57% in the most favourable region for template Rhodotorula mucilaginosa KR, strain Rhodotorula mucilaginosa PY18, and mutant Rhodotorula mucilaginosa E54, respectively. In molecular docking studies, the results of template Rhodotorula mucilaginosa KR endo-PG1 showed an interaction with an affinity score of - 6.0, - 5.9, and - 5.6 kcal/mol for active sites 1, 2, and 3, respectively. Rhodotorula mucilaginosa PY18 endo-PG1 showed an interaction affinity with a score of - 5.8, - 6.0, and - 5.0 kcal/mol for active sites 1, 2, and 3, respectively. Mutant Rhodotorula mucilaginosa E54 endo-PG1 showed an interaction affinity of - 5.6, - 5.5, - 5.5 and - 5.4 kcal/mol for active sites 1, 2, and 3, respectively. The endo-PGI genes of both the yeast strain Rhodotorula mucilaginosa PY18 and mutant Rhodotorula mucilaginosa E54 were successfully cloned and expressed in E. coli DH5α, showing significantly higher endo-PG1 activity, which recorded 94.57 and 153.10 U/mg for recombinant Rhodotorula mucilaginosa pGEM-PGI-PY18 and recombinant mutant Rhotorula pGEM-PGI-E54, respectively.

Space exposure enhanced pectin-degrading enzymes expression and activity in Aspergillus costaricaensis.

Aspergillus is a well-studied fungal genus that is widely used in the processing of plant biomass in industries. This study investigated the effects of space exposure on the ability of Aspergillus costaricaensis, a filamentous fungus isolated from rotten orange peel, to degrade pectin. These fungal spores were carried into space by the Long March 5B carrier rocket and exposed to cosmic radiation for 79 h. After the flight, these spores were resuscitated, and then the growing strains were screened with pectin as the sole carbon source, and the pectinase activity was evaluated. A mutant with increased biomass accumulation ability and pectin-degrading activity compared to the ground control strain was obtained. Comparative transcriptome analysis revealed that several CAZymes genes were significantly upregulated in the mutant, especially those related to pectin degradation. Among the 44 pectinases identified from the annotated genome, 42 were up-regulated. The activities of these pectinases are able to synergistically break down the structure of pectin. In addition, the expression of some genes involved in metabolism, sugar transport, and stress response was altered. These results imply that space exposure might serve as a potential mutagenesis breeding technique, offering the opportunity to acquire biomass-degrading microbial strains with potential for industrial application.

Fungal pectinases: an insight into production, innovations and applications.

The fungal system holds morphological plasticity and metabolic versatility which makes it unique. Fungal habitat ranges from the Arctic region to the fertile mainland, including tropical rainforests, and temperate deserts. They possess a wide range of lifestyles behaving as saprophytic, parasitic, opportunistic, and obligate symbionts. These eukaryotic microbes can survive any living condition and adapt to behave as extremophiles, mesophiles, thermophiles, or even psychrophile organisms. This behaviour has been exploited to yield microbial enzymes which can survive in extreme environments. The cost-effective production, stable catalytic behaviour and ease of genetic manipulation make them prominent sources of several industrially important enzymes. Pectinases are a class of pectin-degrading enzymes that show different mechanisms and substrate specificities to release end products. The pectinase family of enzymes is produced by microbial sources such as bacteria, fungi, actinomycetes, plants, and animals. Fungal pectinases having high specificity for natural sources and higher stabilities and catalytic activities make them promising green catalysts for industrial applications. Pectinases from different microbial sources have been investigated for their industrial applications. However, their relevance in the food and textile industries is remarkable and has been extensively studied. The focus of this review is to provide comprehensive information on the current findings on fungal pectinases targeting diverse sources of fungal strains, their production by fermentation techniques, and a summary of purification strategies. Studies on pectinases regarding innovations comprising bioreactor-based production, immobilization of pectinases, in silico and expression studies, directed evolution, and omics-driven approaches specifically by fungal microbiota have been summarized.

Low temperature inhibits pectin degradation by PpCBFs to prolong peach storage time.

Low-temperature storage is a widely used method for peach fruit storage. However, the impact of PpCBFs on pectin degradation during low-temperature storage is unclear. As such, in this study, we stored the melting-flesh peach cultivar "Fuli" at low temperature (LT, 6°C) and room temperature (RT, 25°C) to determine the effect of different temperatures on its physiological and biochemical changes. Low-temperature storage can inhibit the softening of "Fuli" peaches by maintaining the stability of the cell wall. It was found that the contents of water-soluble pectin and ionic-soluble pectin in peach fruit stored at RT were higher than those stored at LT. The enzyme activities of polygalacturonase (PG), pectate lyase (PL), and pectin methylesterase (PME) were all inhibited by LT. The expressions of PpPME3, PpPL2, and PpPG were closely related to fruit firmness, but PpCBF2 and PpCBF3 showed higher expression levels at LT than RT. The promoters of PpPL2 and PpPG contain the DER motif, which suggested that PpCBF2 and PpCBF3 might negatively regulate their expression by directly binding to their promoters. These results indicated that LT may maintain firmness by activating PpCBFs to repress pectin-degradation-related enzyme genes during storage.

Genome-wide identification and expression analysis of the polygalacturonase gene family in sweetpotato.

BACKGROUND: Polygalacturonase (PG), a crucial enzyme involved in pectin degradation, is associated with various plants' developmental and physiological processes such as seed germination, fruit ripening, fruit softening and plant organ abscission. However, the members of PG gene family in sweetpotato (Ipomoea batatas) have not been extensively identified. RESULTS: In this study, there were 103 PG genes identified in sweetpotato genome, which were phylogenetically clustered into divergent six clades. The gene structure characteristics of each clade were basically conserved. Subsequently, we renamed these PGs according to their locations of the chromosomes. The investigation of collinearity between the PGs in sweetpotato and other four species, contained Arabidopsis thaliana, Solanum lycopersicum, Malus domestica and Ziziphus jujuba, revealed important clues about the potential evolution of the PG family in sweetpotato. Gene duplication analysis showed that IbPGs with collinearity relationships were all derived from segmental duplications, and these genes were under purifying selection. In addition, each promoter region of IbPG proteins contained cis-acting elements related to plant growth and development processes, environmental stress responses and hormone responses. Furthermore, the 103 IbPGs were differentially expressed in various tissues (leaf, stem, proximal end, distal end, root body, root stalk, initiative storage root and fibrous root) and under different abiotic stresses (salt, drought, cold, SA, MeJa and ABA treatment). IbPG038 and IbPG039 were down-regulated with salt, SA and MeJa treatment. According to the further investigation, we found that IbPG006, IbPG034 and IbPG099 had different patterns under the drought and salt stress in fibrous root of sweetpotato, which provided insights into functional differences among these genes. CONCLUSION: A total of 103 IbPGs were identified and classified into six clades from sweetpotato genome. The results of RNA-Seq and qRT-PCR suggested that IbPG006, IbPG034 and IbPG099 might play a significant role in tissue specificity as well as drought and salt stress responses, which showed valuable information for further functional characterization and application of the IbPGs.

Polygalacturonase gene family analysis identifies FcPG12 as a key player in fig (Ficus carica L.) fruit softening.

BACKGROUND: The fig (Ficus carica L.) tree has high economic value. However, its fruit have a short shelf life due to rapid softening. Polygalacturonases (PGs) are essential hydrolases, responsible for the pectin degradation that plays a key role in fruit softening. However, fig PG genes and their regulators have not yet been characterized. RESULTS: In this study, 43 FcPGs were identified in the fig genome. They were non-uniformly distributed on 13 chromosomes, and tandem repeat PG gene clusters were found on chromosomes 4 and 5. Ka/Ks calculation and collinear analysis indicated negative selection as the main driver of FcPG family expansion. Fourteen FcPGs were found expressed in fig fruit with FPKM values > 10, of which seven were positively correlated, and three, negatively correlated with fruit softening. Eleven FcPGs were upregulated and two downregulated in response to ethephon treatment. FcPG12, a member of the tandem repeat cluster on chromosome 4, was selected for further analyses due to its sharp increment in transcript abundance during fruit softening and its response to ethephon treatment. Transient overexpression of FcPG12 led to decreased fig fruit firmness and increased PG enzyme activity in the tissue. Two ethylene response factor (ERF)-binding GCC-box sites were found on the FcPG12 promoter. Yeast one-hybrid and dual luciferase assays showed that FcERF5 binds directly to the FcPG12 promoter and upregulates its expression. Transient overexpression of FcERF5 upregulated FcPG12 expression, thereby increasing PG activity and fruit softening. CONCLUSIONS: Our study identified FcPG12 as a key PG gene in fig fruit softening, and its direct positive regulation by FcERF5. The results provide new information on the molecular regulation of fig fruit softening.

Plant polygalacturonase structures specify enzyme dynamics and processivities to fine-tune cell wall pectins.

Polygalacturonases (PGs) fine-tune pectins to modulate cell wall chemistry and mechanics, impacting plant development. The large number of PGs encoded in plant genomes leads to questions on the diversity and specificity of distinct isozymes. Herein, we report the crystal structures of 2 Arabidopsis thaliana PGs, POLYGALACTURONASE LATERAL ROOT (PGLR), and ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2), which are coexpressed during root development. We first determined the amino acid variations and steric clashes that explain the absence of inhibition of the plant PGs by endogenous PG-inhibiting proteins (PGIPs). Although their beta helix folds are highly similar, PGLR and ADPG2 subsites in the substrate binding groove are occupied by divergent amino acids. By combining molecular dynamic simulations, analysis of enzyme kinetics, and hydrolysis products, we showed that these structural differences translated into distinct enzyme-substrate dynamics and enzyme processivities: ADPG2 showed greater substrate fluctuations with hydrolysis products, oligogalacturonides (OGs), with a degree of polymerization (DP) of ≤4, while the DP of OGs generated by PGLR was between 5 and 9. Using the Arabidopsis root as a developmental model, exogenous application of purified enzymes showed that the highly processive ADPG2 had major effects on both root cell elongation and cell adhesion. This work highlights the importance of PG processivity on pectin degradation regulating plant development.

Fine mapping and gene silencing pinpoint Capana10g002229 as a strong candidate gene regulating the deciduous character of ripe pepper fruit (Capsicum spp.).

KEY MESSAGE: The pepper S locus, which controls the deciduous character of ripe fruit, was first fine mapped into an interval with a physical length of ~ 38.03 kb on chromosome P10. Capana10g002229, encoding a polygalacturonase, was proposed as a strong candidate gene based on sequence comparison, expression pattern analysis and virus-induced gene silencing (VIGS). The deciduous character of ripe fruit, which is controlled by the dominant S locus, is a domesticated trait with potential value in the pepper processing industry (Capsicum spp.). However, the gene associated with the S locus has not been identified. Here, one major QTL designated S10.1 was detected by using the F2 population (n = 155) derived from BA3 (Capsicum annuum) × YNXML (Capsicum frutescens) and was further verified in an intraspecific backcross population (n = 254) derived from the cross between BB3 (C. annuum) and its wild relative Chiltepin (C. annuum var. glabriusculum) with BB3 as the recurrent parent. Then, a large BC1F2 population derived from the self-pollination of BB3 × (BB3 × Chiltepin) individuals and comprising 4217 individuals was used to screen the recombinants, and the S locus was ultimately delimited into a 38.03-kb region on chromosome P10 harbouring four annotated genes. Capana10g002229, encoding a polygalacturonase (PG), was proposed as the best candidate gene for S based on sequence comparison and expression pattern analyses. Downregulation of Capana10g002229 in fruits through VIGS significantly delayed fruit softening and abscission from the fruit-receptacle junction. Taken together, the results show that Capana10g002229 could be regarded as a strong candidate gene associated with the S locus in pepper. These findings not only lay a foundation for deciphering the molecular mechanisms underlying pepper domestication but also provide a strategy for genetic improvement of the deciduous character of ripe fruit using a marker-assisted selection approach.

Pectinase from a Fish Gut Bacterium, Aeromonas guangheii (SS6): Production, Cloning and Characterization.

During the present research, 11 gut bacteria were isolated from the freshwater fish, Systomus sarana (General name: olive barb) and upon screening, the strains produced extracellular pectinase enzyme. Among them, the SS6 strain was found to produce a high quantity of 208.731 U/ml pectinase and through molecular characterization the SS6 strain was identified as Aeromonas guangheii. During the culture of SS6 strain, a set of parameters were optimized through the response surface methodology with a Box-Behnken design, for the production of the enzyme. The optimal conditions were found to be 2.11% of maltose, 2.20% of yeast extract, 6.5 of pH, and a temperature of 27.3 °C at 32-h incubation. Under the above conditions, the activity of pectinase production was enhanced to 371 U/ml. The purified pectinase's molecular weight was determined to be ~ 50 kDa (by 10% 2-D PAGE). Totally, nine peptides were identified from the purified pectinase enzyme through the MALDI-TOF-MS analysis and MASCOT tool was used to get the mass spectrum of the peak at 2211 of peptide that indicated the reference pectinase protein. The referenced gene primer (pectate lyases) was PCR amplified and its nucleotide sequence was analyzed. The exo-pelA gene was cloned in pREST vector, which was found to be over expressed in Escherichia coli BL21. The ORF encoded for a mature protein comprising of 425 amino acids (1236 nucleotides) with a predicted molecular weight of ~ 48.7 kDa. The present findings underline the potential of the fish-gut microbes as a source of biotechnologically important enzymes.

New Saccharomyces cerevisiae-Kluyveromyces marxianus fusant shows enhanced alcoholic fermentation performance.

Kluyveromyces marxianus has a promising enological potential because of strong extracellular pectinase activity and the copious production of specific higher alcohols and esters. However, K. marxianus is unable to complete alcoholic fermentation on its own. For this reason it is only used in co- or sequential fermentation in conjunction with Saccharomyces spp. Here we applied the protoplast fusion technique to two commercial Saccharomyces cerevisiae strains (Lalvin EC1118® and Lalvin QA23®) and K. marxianus isolate IWBT Y885, to obtain fusants that possess pre-selected, enologically relevant features of K. marxianus, but with improved fermentation performance. After an extensive selection process, performed using non-auxotrophic markers, we identified one fusant, BF2020, that shows enhanced fermentation performance compared to the parental strain K. marxianus Y885, but that still exhibits strong pectinase activity and may be suitable for industrial production. The genome of the selected hybrid was further investigated and characterized by RAPD-PCR analysis and whole genome sequencing.

Metabolic novelty originating from horizontal gene transfer is essential for leaf beetle survival.

Horizontal gene transfer (HGT) provides an evolutionary shortcut for recipient organisms to gain novel functions. Although reports of HGT in higher eukaryotes are rapidly accumulating, in most cases the evolutionary trajectory, metabolic integration, and ecological relevance of acquired genes remain unclear. Plant cell wall degradation by HGT-derived enzymes is widespread in herbivorous insect lineages. Pectin is an abundant polysaccharide in the walls of growing parts of plants. We investigated the significance of horizontally acquired pectin-digesting polygalacturonases (PGs) of the leaf beetle Phaedon cochleariae. Using a CRISPR/Cas9-guided gene knockout approach, we generated a triple knockout and a quadruple PG-null mutant in order to investigate the enzymatic, biological, and ecological effects. We found that pectin-digestion 1) is exclusively linked to the horizontally acquired PGs from fungi, 2) became fixed in the host genome by gene duplication leading to functional redundancy, 3) compensates for nutrient-poor diet by making the nutritious cell contents more accessible, and 4) facilitates the beetles development and survival. Our analysis highlights the selective advantage PGs provide to herbivorous insects and demonstrate the impact of HGT on the evolutionary success of leaf-feeding beetles, major contributors to species diversity.

VmMon1-Ccz1 Complex Is Required for Conidiation, Autophagy, and Virulence in Valsa mali.

Apple Valsa canker caused by Valsa mali is a serious disease in eastern Asia, especially in China. In our previous proteomics study, monensin sensitivity 1 protein in Valsa mali (VmMon1) was identified to be significantly upregulated during V. mali infection. It was reported Mon1 protein formed a heterodimer called MC (Mon1-Ccz1) complex with caffeine, calcium, and zinc sensitivity 1 protein (Ccz1) in yeast. However, Ccz1 had not been identified in plant-pathogenic fungi such as Fusarium graminearum and Magnaporthe oryzae. Here, we identified a Ccz1 ortholog VmCcz1 in V. mali, by using DELTA-BLAST. The interaction of VmMon1 and VmCcz1 were verified using yeast two-hybrid assay, bimolecular fluorescence complementation, and co-immunoprecipitation assays. Further yeast three-hybrid screenings determined that VmRab7 (Ras-related protein in V. mali) interacted with the MC complex. Targeted gene deletion showed that the ∆VmMon1 and ∆VmCcz1 mutants were defective in vegetative growth, conidiation, and pathogenicity. In addition, both mutants were more sensitive to osmotic and oxidative stresses and intracellular protein transport inhibitors. Cytological examination revealed that the ∆VmMon1 and ∆VmCcz1 mutants were impaired in vacuole fusion and autophagy. More importantly, expression of pectinase genes decreased in both mutants compared with those of the wild type during infection. Overall, our study identified Mon1 and Ccz1 genes in V. mali and provided evidence that VmMon1 and VmCcz1 are critical components that modulate vacuole fusion and autophagy, thereby affecting the development, conidiation, and pathogenicity of V. mali. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

SlMBP3 Knockout/down in Tomato: Normal-Sized Fruit with Increased Dry Matter Content through Non-Liquefied Locular Tissue by Altered Cell Wall Formation.

The phenotypic effect of the knockdown/out of AGAMOUS clade MADS-box gene SlMBP3 in tomato was evaluated using a transferred DNA (T-DNA)-tagged mutant of SlMBP3 and SlMBP3-RNA interference lines. SlMBP3 was preferentially expressed in the locular tissue of fruit and the seed coat combined with the endoderm. Consistent with where SlMBP3 is expressed, the SlMBP3-knockout/down lines showed non-liquefied locular tissues and increased number of seed hairs than the wild type (WT). The early cell degradation of the locular tissue was not observed in the fruits of the SlMBP3-knockout/down lines, and the cells were elongated like placental cells resulting in non-liquefied locular tissues. As the result, the fruits of the SlMBP3-knockout/down lines exhibited higher dry matter contents and titratable acidity than those of the WT. During locular tissue cell development under the SlMBP3 knockout/down, the expression of cell-enlargement-related genes (beta-expansin gene SlEXPB1 and endo-beta-1,4-D-glucanase gene Cel8) and pectinase-inhibitor-related genes (pectin esterase inhibitor gene PE inhibitor and polygalacturonase inhibitor gene PG inhibitor) was upregulated and that of pectinase-encoding genes (polygalacturonase gene QRT3-like and pectin lyase gene PL2) was downregulated. In the seed coat of the SlMBP3-knockout/down lines, tomato trichome-formation-related genes such as MYB genes containing R2 and R3 repeats (R2R3-MYB) transcription factor SlMYB75, B-type cyclin SlCycB2 and Homeodomain Leucine Zipper (HD-Zip) IV transcription factor Woolly were downregulated. Our results demonstrate that SlMBP3 is involved in the liquefaction of the locular tissue through the modification of cell development and degradation processes and seed hair formation in tomato fruits, and the SlMBP3 knockout/down results in normal-sized fruit with increased dry matter content.

Tandemly duplicated genes encoding polygalacturonase inhibitors are associated with bruchid (Callosobruchus chinensis) resistance in moth bean (Vigna aconitifolia).

Bruchids are stored-grain insect pests responsible for serious seed loss in legume crops. A previous study using an F2 population (F2OA) derived from a cross between wild moth-bean (Vigna aconitifolia [Jacq.] Maréchal) accession TN67 (resistant) and cultivated moth-bean accession ICPMO056 (susceptible) revealed that resistance to the azuki bean weevil (Callosobruchus chinensis L.) in TN67 was regulated by a single gene located in the major quantitative trait locus-qVacBrc2.1. In this study, qVacBrc2.1 was finely mapped and candidate genes in this locus were identified using F2OA and another large F2 population (F2NB) derived from the cross mentioned previously. In contrast to the previous study, segregation analysis in the F2NB population revealed that resistance against this pest was controlled by two genes. Furthermore, the addition of novel markers to qVacBrc2.1 and reanalysis of the QTL in the F2OA population demonstrated that qVacBrc2.1 constituted two linked QTLs-qVacBrc2.1-A and qVacBrc2.1-B. The presence of qVacBrc2.1-B was verified using the population F2NB. Comparative genomics using three Vigna spp. strongly suggested the presence of two tandemly duplicated genes, VacPGIP1 and VacPGIP2, which encoded polygalacturonase inhibitors (polygalacturonase-inhibiting proteins) as the candidates for conferring resistance, but only VacPGIP1 could be successfully cloned and sequenced. The alignment of VacPGIP1 coding sequences of TN67 and ICPMO056 revealed eight single nucleotide polymorphisms, three of which altered the amino-acid sequence of the predicted domains of polygalacturonase inhibitors in ICPMO056. Overall, these findings indicate that VacPGIP1 and VacPGIP2 regulated C. chinensis resistance in TN67.

Pectinolytic arsenal of Colletotrichum lindemuthianum and other fungi with different lifestyles.

AIM: To identify and analyse genes that encode pectinases in the genome of the fungus Colletotrichum lindemuthianum, evaluate the expression of these genes, and compare putative pectinases found in C. lindemuthianum with pectinases produced by other fungi and oomycetes with different lifestyles. METHODS AND RESULTS: Genes encoding pectinases in the genome of C. lindemuthianum were identified and analysed. The expression of these genes was analysed. Pectinases from C. lindemuthianum were compared with pectinases from other fungi that have different lifestyles, and the pectinase activity in some of these fungi was quantified. Fifty-eight genes encoding pectinases were identified in C. lindemuthianum. At least six types of enzymes involved in pectin degradation were identified, with pectate lyases and polygalacturonases being the most abundant. Twenty-seven genes encoding pectinases were differentially expressed at some point in C. lindemuthianum during their interactions with their host. For each type of pectinase, there were at least three isoenzyme groups. The number of pectinases present in fungi with different lifestyles seemed to be related more to the lifestyle than to the taxonomic relationship between them. Only phytopathogenic fungi showed pectate lyase activity. CONCLUSIONS: The collective results demonstrate the pectinolytic arsenal of C. lindemuthianum, with many and diverse genes encoding pectinases more than that found in other phytopathogens, which suggests that at least part of these pectinases must be important for the pathogenicity of the fungus C. lindemuthianum. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of these pectinases could further the understanding of the importance of this broad pectinolytic arsenal in the common bean infection and could be exploited for biotechnological purposes.