De novo assembly and comparative transcriptome analysis of Monilinia fructicola, Monilinia laxa and Monilinia fructigena, the causal agents of brown rot on stone fruits

- We sequenced, assembled and annotated the transcriptomes of the three pathogens using mRNA from germinating conidia and actively growing mycelia of two isolates of opposite mating types per each species for comparative transcriptome analyses..
- Approximately, 50% of the assembled contigs had significant hits when blasted against the NCBI non-redundant protein database and top-hits results were represented by Botrytis cinerea, Sclerotinia sclerotiorum and Sclerotinia borealis proteins..
- More than 90% of the obtained sequences were complete, the percentage of duplications was always less than 14% and fragmented and missing transcripts less than 5%.
- Conclusions: This is the first large-scale reconstruction and annotation of the complete transcriptomes of M..
- 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0.
- 3, per litre) in the dark for 4 days;.
- D = mycelium grown in the dark for 4 days.
- L = mycelium grown in the dark for 2 days and then exposed to light for 2 days.
- index.php?page=trimmomatic) setting the parameters as follows: i) LEADING and TRAILING = 3, removing bases from the two ends of the reads if below a threshold quality of 3.
- trinityrnaseq/wiki) was used for the de novo assembly of the transcriptomes using together sequencing data from the six libraries (2 isolates and 3 growing conditions) from each species (Table 1).
- Default assembly parameters of Trinity were used, with the addition of the “–jaccard_clip”.
- The annotation of the putative transcripts obtained from each assembly was performed using local BLAST+ 2.3.0 (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast.
- tBLASTn search was used to identify orthologous tran- scripts in the three Monilinia species.
- More than 80% of the paired-end reads used for the assembly successfully mapped to the respective as- sembled transcriptome used as reference (Additional file 1: Table S2)..
- Most of them (84% for MFRC, 83% for MLAX, 79% for MFRG) displayed a coverage higher than 70% of the protein length.
- Data obtained from transcriptome completeness analysis by BUSCO, based only on conserved fungal orthologs, showed that over 90% of the assembled tran- scripts for the three Monilinia species were complete and that the percentage of duplicated transcripts was always less than 14%.
- More than 50% of the assembled contigs had a significant hit in BLASTx search (Table 4).
- The E-value distribution of the top blast hits showed about 50% of hits with an E-value equal to zero and about 70% with E-values ranging from 0 to 1e-61 (Fig.
- In the three transcriptomes, more than 80% of the uni- genes with a significant hit were associated to at least one GO term, and the GO terms were functionally classified and plotted.
- In total 2536 (MFRC), 2734 (MLAX) and 2753 (MFRG) unigenes (17–23% of the genes with BLAST hits) were assigned to 24 functional categories of clusters of OGs (Fig.
- The number of uni- genes enclosed in the OG clusters “function unknown” in MFRG and “translation, ribosomal structure and biogenesis”.
- cinerea as reference, whereas 407 transcripts were spe- cific for at least one of the Monilinia species and had not homologous sequences in B.
- Heat-map representation of expression profiles of the DETs from the two isolates per species under the differ- ent growing conditions confirmed the diversity among.
- Twelve DETs encoding hydrolytic or carbohydrate-active enzymes were over-expressed in MFRC, including glyco- side hydrolases (GHs) such as a β-1,3-glucan binding protein of the GH16 family (T9687.
- Full-length or partial DETs annotated as α/β hydrolases were over-expressed (T9625, T9489 and T9732) or uniquely assembled (T10098) in MFRC.
- Nine DETs of the same enzymatic group were over-expressed in MLAX including a putative ß-1,6-galactanase protein (T309.
- Four DETs were over-expressed in MFRG, including T7115 (FC encoding a putative α-amylase (GH13 family), and T6223 (FC encoding a ß-mannosidase B-like protein (GH2 family).
- Three DETs were over-expressed in MFRC.
- T9914 (FC encodes a secreted trypsin-like serine protease with homologs in the closely related species B.
- Six DETs were significantly over-expressed in MFRC..
- FC a putative fatty acid elongase (FEN1) of the GNS1/SUR4 family like protein (T9722.
- A putative glycosylphosphatidylinositol-anchored pro- tein (GPI-AP) was significantly over-expressed in MLAX (T205.
- The DETs over-expressed in MFRC included two transcrip- tion factors containing a N-terminal GAL4-like (Zn 2 Cys 6 ) DNA-binding domain (GAL4 TF), T9760 (FC and T9765 (FC .
- A regulator of G protein signalling (RGS) (T312) was over-expressed in MLAX (FC as well as a protein kinase-like sequence (T321.
- NH = No homologs in the B.
- Over-expressed transcripts are highlighted in bold.
- A Zn finger C2H2 TF (T63) was over-expressed in MFRG (FC = 11.7), with no orthologs in MFRC.
- Twelve DETs were over-expressed or unique in MFRC..
- Additional MFSs were over-expressed (T9642, T9594 and T9657) or exclusive (T10106 and T10002).
- DETs included an oligopeptide transporter of the OPT family (T9643) and a potassium uptake protein (T9629) (FC .
- They in- cluded, in the order: a serine hydrolase (FSH1) domain-containing protein (T9586).
- A putative phytanoyl-CoA dioxygenase (PhyH) family protein (T9539), significantly over-expressed in MFRC (FC = 9.4), with homologs in S.
- A short-chain dehydrogenase/reductase displaying a cla- vulanic acid dehydrogenase, classical (c) SDR domain (T253) was significantly over-expressed in MLAX (FC.
- Orthologues of the BcNRPS1, coded T8874, were fragmented in MLAX and over-expressed in MFRG (FC like T6684 (FC which encodes a protein sharing homology with a FAD-binding-domain containing protein of S.
- were sig- nificantly over-expressed in MFRC (FC .
- id up to 45%) were over-expressed in.
- was over-expressed in MFRG (FC .
- A zinc ion binding protein (T9754) containing a WLM (WSS1-like metalloprotease) domain was over-expressed in MFRC (FC .
- In detail, T10367 and T10387 encode polyproteins with conserved domains of reverse transcriptase (RT) and ribo- nuclease H (RNase H) enzymes from long terminal repeats (LTRs) retroelements of the Ty3/Gypsy family, sharing homology with proteins from B.
- T10350 encodes a polyprotein with RT and RNase H domains from LTRs retroelements of the Ty1/.
- id = 63%) of the Long INterspersed Elements (LINE) class.
- T5170 encodes a transposase of the DDE super- family endonuclease with homologs in B.
- Among DETs over-expressed in MFRC we found an al- lergen protein (T9817.
- T10278, fully assembled in MLAX and partially in MFRG, encodes a putative ATPase of the AAA family..
- The GHs over-expressed in MFRC included a β-1,3-glucan binding protein that in Phanerochaete chry- sosporium is involved in the degradation of laminarin [27].
- The DETs over-expressed in MLAX included plant cell-wall degrading enzymes, such as a pectin methylester- ase, a ß-1,6-galactanase protein (GH5), a xyloglucan-specific endo-ß-1,4-glucanase (GH12) and an α -1,2-mannosidase (GH92).
- GHs over-expressed in MFRG include a putative α-amylase (GH13) acting on α-glycosidic bonds and involved in lignocellulose saccharification processes [36] and a ß-mannosidase B-like protein (GH2), enzymes displaying different substrate specificity in Aspergillus spp.
- A protease inhibitor containing a PEBP domain was over-expressed in MFRC.
- This class of proteins in- cludes secreted bacterial insecticidal and nematocidal toxins and intercellular signalling proteins mediating bacterial-host or bacterial-bacterial interactions (e.g., [42]) A different class of the same family includes the Ss-RHS1 secretory protein from S.
- A trypsin-like serine protease was over-expressed in MFRC while a secreted pepsin-like aspartic proteases was over-expressed in MLAX..
- Among DETs related to fungal morphogenesis and de- velopment, we found over-expressed in MFRC a developmental-specific protein homolog to the S.
- Other DETs were over-expressed in MFRC:.
- DETs over-expressed in MLAX include a GPI anchored protein, important for cell wall synthesis and integrity in N.
- and a putative ATPase of the AAA family, a large and diverse group of enzymes inducing conformational changes in a wide range of pro- teins associated with cell-cycle regulation, proteolysis, organelle biogenesis and intracellular transport [55]..
- A Zn finger C2H2 TF was over-expressed in MFRG..
- and a putative signal sequence protein of the Tat secretion pathway that serves to actively translocate fully folded proteins across membranes [63]..
- Bifunctional solanapyrone synthases, over-expressed in both MFRC and MFRG, are involved in the biosynthesis of the polyketide-derived phytotoxins solanapyrones in Alternaria solani, Ascochyta rabiei and other fungi.
- Enzymes of this family are involved in the degradation pathway of aromatic com- pounds and in the biosynthesis of the β -lactam antibiotic penicillin G in P.
- Several DETs are involved in the biosynthesis of clavulanic acid, a potent inhibitor of bacterial class A serine β-lactamases, with weak antibiotic activity.
- with a CAS-like protein of the human fungal pathogen Glarea lozoyensis.
- a putative CAD protein was over-expressed in MLAX.
- Moreover, a PhyH protein coded by the thnG gene from Streptomyces cattleya is involved in the biosynthesis of the β-lactam antibiotic thienamycin [70]..
- In MFRC a putative enoyl-hydratase isomerase contain- ing a transferase domain showed similarity with the fumigaclavine B O-acetyltransferase involved in the bio- synthesis of fumigaclavine C, an ergot alkaloid produced by fungi of the Trichocomaceae family [71] and with the trichothecene 3-O-acetyltransferases from Fusarium spp., which modify the trichothecene mycotoxin deoxy- nivalenol (DON) and reduce its toxicity [72], while a cytochrome P450 monooxygenase is related to isotricho- dermin C-15 hydroxylases involved in the biosynthesis of trichothecenes [73].
- DETs identified in MLAX in- cluded a benzoate 4-monooxygenase cytochrome P450 protein homologous to fungal trichodiene oxygenases, like the Fusarium sporotrichioides Tri4, involved in the trichothecene biosynthesis [74], and a cytochrome P450 monooxygenase showing similarity to enzymes essential for the synthesis of the polyketide-derived mycotoxin sterigmatocystin [75].
- are involved in the biosynthesis of the trichothecene and gliotoxin [76]..
- Several transcripts related to LTR retroelements of the Ty3/Gypsy and the Ty1/Copia families were gener- ally identified as unique in MFRG, revealing an active retrotransposition in the pathogen genome..
- In this study, we provide the first large-scale recon- struction and annotation of the transcriptomes of the phytopathogenic fungi MFRC, MLAX and MFRG which are the main causes of heavy yield losses on pome and stone fruits all around the world.
- The transcrip- tomes of the three Monilinia species did not show any significant differences in the GO and OG cluster functional categories.
- Consequently, orthologous tran- scripts were identified in the three species on the base of the B.
- cinerea proteome and, additionally, more than 400 transcripts specific for at least one of the Monilinia species with no homologs in the refer- ence were detected.
- A comparative analysis among orthologs in the three species based on transcript abundance revealed DETs over-expressed or exclusive for each of the three species.
- Moreover, MFRC was characterised by a very high expression of the developmental-specific protein Ssp1, cell surface proteins acting as virulence factors, enzymes responsible for oxylipin production and regulation of spore differentiation and plant-pathogen interactions, a MSS4-like protein playing a role in apical hyphal growth, numerous MFS transporters related to pathogenesis and multidrug resistance, enzymes involved in the biosyn- thesis of antimicrobials (solanapyrone, clavulanic acid) and mycotoxins, and β-lactamases involved in anti- microbial resistance.
- The data obtained represent new insights in the transcriptome analyses of the three species of Monilinia and provide a new and comprehensive genetic resource that will contribute to get deeper knowledge on the population biology, physiology and plant-pathogen interactions of these important phytopathogenic fungi which are essential for improving sustainable crop pro- tection strategies..
- Bioinformatic analysis was partially carried out by using the facilities of the ReCaS data center of the University of Bari (www.recas-bari.it)..
- The research was partially carried out in the framework of the project.
- The funding company had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript..
- FF designed the experiments, supervised and complemented the writing and coordinated the collaboration of the authors.
- The Monilinia isolates used in this study were stored in the fungal collection of the Department of Soil, Plant and Food Sciences - Plant Pathology Section of the University of Bari (Italy).
- No permits were required for the collection of the fungal samples..
- Variations in the molecular and physiological characteristics and the virulence of Monilinia fructicola, M.
- SSR marker analysis of Monilinia fructicola from Swiss apricots suggests introduction of the pathogen from neighbouring countries and the United States.
- Characterization of the cytochrome b (cyt b) gene from Monilinia species causing brown rot of stone and pome fruit and its significance in the development of QoI resistance.
- Overexpression of a redox-regulated cutinase gene, MfCUT1, increases virulence of the brown rot pathogen Monilinia fructicola on Prunus spp.
- Mating system in the brown rot pathogens Monilinia fructicola, Monilinia laxa and Monilinia fructigena.
- Prediction driven functional annotation of hypothetical proteins in the major facilitator superfamily of S.
- Changes in the Sclerotinia sclerotiorum transcriptome during infection of Brassica napus.
- Mutational analysis of the glycosylphosphatidylinositol (GPI) anchor pathway demonstrates that GPI-anchored proteins are required for cell wall biogenesis and normal hyphal growth in Neurospora crassa.
- Essential letters in the fungal alphabet.
- Production of the antibiotic secondary metabolite solanapyrone a by the fungal plant pathogen Ascochyta rabiei during fruiting body formation in saprobic growth.
- Mutational analysis of the thienamycin biosynthetic gene cluster from Streptomyces cattleya

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