- Background: Laurel wilt caused by Raffaelea lauricola is a lethal vascular disease of North American members of the Lauraceae plant family. - In the ensuing time, laurel wilt has caused and continues to cause widespread mortality on redbay (Persea borbonia) and other members of the Lauraceae family in the southeastern USA resulting in massive eco- system damage [6–11]. - Full list of author information is available at the end of the article. - In the southeastern USA where laurel wilt has annihilated native Lauraceae trees, invasive camphor trees from Asia (Cinnamomum camphora) exhibit limited symptoms in- cluding branch dieback or no symptoms of laurel wilt unless subject to mass beetle attacks [19]. - lauricola in the USA has been assumed to be from a single introduction [17]. - [22] have examined North American and Asian isolates and further narrowed the origin of the North American population to Taiwan. - lauricola is so aggressive that a single inocu- lation of this pathogen on avocado and other members of the North American Lauraceae family is sufficient to induce systemic and lethal disease development within several weeks [27, 29] with as few as 100 spores [30].. - Chitin, a major structural component of fungal cell walls, is one of the best-characterized fungal PAMPs. - lauricola is a non-pathogen in trees native to southeast Asia and the lethal symptomology observed in Lauraceae hosts native to the western hemisphere is the result of a massive defense response of the previously un-encountered host species sensing the presence of a potential pathogen within the xylem tissue associated with beetle galleries.. - A second hypothesis which is truly an “evolutionary mismatch hypothesis” in the con- text of Desurmont et al. - To test and distinguish between these two hy- potheses and better understand the adaptations present in pathogenic relative to non-pathogenic Raffaelea spp., we generated, annotated, and compared high-quality draft genome assemblies of the pathogenic R. - To conduct comparative genomics analyses between pathogenic and non-pathogenic Raffaelea spp., we se- quenced and assembled genomes and transcriptomes of the pathogenic species R. - A total of 8.64% of the R. - The increased repeat content of the R. - lauricola genome could be attributed primarily to an increase in LTR retroelements which comprised 2% of the R. - of the R. - LINE retroelements and DNA transposons comprised 0.6 and 1% of the R. - MAKER predicts proteins based on RNA-Seq transcripts and homology with protein-coding sequences of other species, and with the consensus of the ab initio gene prediction algorithms GeneMark [44], AUGUSTUS [45], and SNAP [46]. - The details of the Maker pipeline can be found in the Methods section. - lauricola, 49 of the 740 secreted proteins are predicted to be effectors (6.6%. - aguacate, 30 of the 727 secreted proteins are predicted to be effectors (4.1%. - Of the 49 pre- dicted effectors from R. - Between the two genomes, 8128 putatively orthologous pairs were identified repre- senting 79 and 70% of the predicted proteomes of R.. - The three dimensional model of the predicted RL4_JR_05745 protein and the crystal structure model of Sm1 (3M3A) are shown in Fig. - The overlay of the Sm1 crystal structure and the RL4_. - aguacate is a member of the Hce2 family homologous to the Cladosporium fulvum (Passalora fulvum) Ecp2 [55].. - a Predicted tertiary structure model of the RL4_JR_05745 protein. - d Predicted tertiary structure model of the full length RL4_JR_08480 protein. - lauricola protein confirmed a struc- ture consistent with numerous aerolysin-like proteins known to function as toxins or defensive molecules by specific sugar binding through the lectin domain and membrane pore-forming activity of the natterin-like do- mains of homo-oligomers. - matched hypothetical proteins in the GenBank non- redundant protein sequence database. - Of the four unique CFEM proteins one was down-regulated during plant infection and the other three were not differentially expressed between culture and infected plant. - These include two homologs of the Pyricularia oryzae ‘biotrophy associated secreted 2’ proteins (RL4_JR_02461 and RL4_JR_02274. - 3.2 and 6.7 log2 fold change, respectively), three homologs of the P. - Seven CAZyme domains were found to be expanded in the non-pathogenic R.. - 3 Comparison of the number of Carbohydrate-Active Enzyme (CAZyme) modules across the R. - Secondary metabolism gene clusters are expanded in the R. - For this reason, the SMCs of the two Raffaelea genomes were predicted using two independent programs, SMURF [58] and the anti- SMASH webserver [59]. - Due to the differences in the al- gorithms used by SMURF and antiSMASH, the two programs identified overlapping but not identical SMC genes. - Layering transcriptomic data onto the SMC data de- termined that 10 of the R. - cluster 7 (NRPS) and cluster 24 (PKS-NRPS) are not found in the R. - A comparison of the genomic regions up and downstream of this missing cluster in the R. - aguacate genome or present in dispersed, non-clustered regions of the genome (Fig. - Cluster 7 represents an even more extreme example of an inser- tion or deletion event in which not only were the eight genes predicted for Cluster 7 missing from the syntenic region of the R. - aguacate genome, but also an additional 11 genes up- and five genes down-stream of the cluster were absent (Fig. - This cluster is notable for the presence not only of the anchoring NRPS but also a fatty acid synthase (acyl-synthetase) and a second NRPS (“HC-toxin synthetase”).. - Of the remaining in planta differentially expressed key enzyme clusters, Clusters 16 and 17 appear to encode siderophores (in addition to Clusters 6 and 9 which are not significantly expressed in planta) and Cluster 8 ap- pears to encode at least a partial dihydroxynapthelene- based melanin biosynthetic pathway (a PKS with high homology to other melanin PKSs and a clustered puta- tive tetrahydroxynaphthalene reductase). - To confirm the host responses of the two redbay genotypes, laurel wilt disease scores were assessed following trunk inoculation. - When mapping reads of the 21 RNAseq samples (6 water-inoculated stems, 6 R. - The per- centage of fungal reads in the plant inoculated samples ranged from to of the total read pairs (Supplemental Table S12). - Taking a more global view of the data, we see that within the top 100 differentially regulated genes (sorted adjusted P-value), 44 are alterna- tive sulfur source (sulfides, sulfoxides, sulfones, sulfo- nates, sulfate esters and sulfamates) uptake or assimilation related genes (Fig. - All seven of the high affinity methionine permeases (TCDFM: 2.A.3.8.4) and all three of the high affinity cysteine permeases (TCDFM:. - When comparing the relative abundance of the TauD domain among other Sordariomycetes species, Neurospora crassa encodes 13 TauD family members, Podospora anserine 23, Colletotrichum graminicola 18, C. - The Cys-3 ortholog was identified by BLASTP homology and RSD analysis of the R. - An ortholog of the Scon-2 negative regulator (RL4_JR_01708) was also identified from the predicted gene calls. - Pathogenicity traits may allow these fungi to survive and proliferate in the unique ecological niche of the living trees’ xylem vessels by initially overcoming or avoiding the host immune system. - When introduced to new, closely-related but not sympatrically co-evolved hosts, this balance of attack and defense is tilted in favor of the pathogen with its novel virulence mechanisms and a host population lacking the corresponding counter defense mechanisms. - lauricola genes have been identified in support of this hypothesis and provide a starting point to understand how the fungus induces symptoms and poses such a serious threat to avocado, redbay and other members of the Lauraceae family in the western hemi- sphere. - The current work develops these genomic resources further by providing gene calls from the new assembly, provid- ing detailed annotation of comparative gene content be- tween the two species, and analyzing the transcript accumulation dynamics of the R. - Comparison of predicted secretomes of the two Raf- faelea species indicated the presence of secreted protein genes unique to R. - In addition to other characterizations of the R. - The lectin do- mains of aerolysin-like proteins have been demonstrated to provide binding specificity within lipid membranes triggering oligomerization of the natterin domain which inserts into the lipid bilayer to allow electrolyte leakage. - Three characteristics of the R.. - However, the Ecp2 effector of Mycosphaerella fijiensis, causal agent of the devastating black Sigatoka disease of banana, causes host necrosis and promotes virulence much stronger than the C. - Characterization of total repeat content of the two genomes indicated that LTR retroelements are ten-fold more abundant in the R. - The rela- tive low abundance of fungal transcripts present in the sampled tissue as well as the limited sampling of the interaction may account for the inability to get an accur- ate assessment of differential gene expression for all clusters. - This lack of resolution is inherent to the inter- action in which the pathogen is detected at low biomass and the large woody nature of the host makes it difficult to spatially sample the host-pathogen interaction. - Several plant chitin receptors located in the plasma membrane have been identified. - In the first group LysMs are associated with chiti- nase domains (GH18). - lauricola LysM effectors have du- plicated and diversified to avoid host immunity responses to a point where the fungus may deploy virulence factors (e.g., cerato-platanin, aerolysin-like protein, Hce2) when it is spatially and temporally poised to take advantage of the host resources. - In addition to the secretion of proteins and metabo- lites for establishing compatibility and promoting dis- ease, pathogens must also adapt their metabolism to that of the host environment. - Although avocado fruit are rich in sulfur, primarily glutathione, the sulfur makeup in other organs of the plant are not well characterized. - The extreme up-regulation of the alternative sulfur. - This hypothesis is being pursued through characterization of the R. - laur- icola Cys-3 homolog which is known to function as a positive global regulator of the alternative sulfur assimi- lation pathways in Ascomycota fungi.. - Due to the lack of arms race co-evolution of the invasive R.. - Single-end sequencing of the genomes of R. - In the first cycle of Maker, only GeneMark-ES v.4.32 was used. - To assess the validity of the final assembly and gene prediction, Benchmarking. - Hmmsearch in the HMMER3 package was used to identify Raffaelea pro- teins containing the Hce2 domain (Pfam: PF14856).. - structure of the Trichodema virens Sm1protein (DOI:. - Approximately 10 ml of sterilized water was added to the surface of the plates and a spreader was used to agitate the surface gently.. - For in vitro cultures utilized in the RNA-Seq analysis, a spore suspension of R. - For redbay trunk inoculations, two 2-mm-diameter holes, 1 cm apart, were drilled into each side of the main stem (7.5 mm deep) at a 45° angle, 15–30 cm above the soil line. - The tree inocula- tion and laurel wilt disease scoring experiment was repeated once and the ranking of the HIE genotype as susceptible and the HIL genotype as tolerant was confirmed.. - For in vitro-grown fungal tissue, approximately 10 mg of the freeze-dried fungal tissue for each sample was ho- mogenized using a bead beater. - Paired- end, 2 × 100 cycle sequencing was performed at the ICBR on two lanes of the Illumina HiSeq3000 instru- ment using the clustering and sequencing reagents provided by Illumina (San Diego, CA, USA).. - Trinity [41] with the default parameters was used to obtain the transcriptome for each of the two redbay genotypes. - Disease scores of the inoculated redbay trees at 60 days post inoculation. - The funding body played no role in the design of the study, the collection, analysis, and interpretation of data or in the writing of the manuscript.. - Exotic bark- and wood-boring Coleoptera in the United States:. - A fungal Symbiont of the Redbay Ambrosia beetle causes a lethal wilt in Redbay and other Lauraceae in the southeastern United States. - A comparison of arborescent vegetation pre- (1983) and post- (2008) outbreak of the invasive species the Asian ambrosia beetle Xyleborus glabratus in a Florida maritime hammock. - Genetic variation in native populations of the Laurel wilt pathogen, Raffaelea lauricola, in Taiwan and Japan and the introduced population in the United States. - Genetic analyses of the Laurel wilt pathogen, Raffaelea lauricola, in Asia provide clues on the source of the clone that is responsible for the current USA epidemic.. - Laurel wilt, caused by Raffaelea lauricola, is detected for the first time outside of the southeastern United States. - Genomic comparisons of the laurel wilt pathogen, Raffaelea lauricola, and related tree pathogens highlight an arsenal of pathogenicity related genes. - Distribution and bioinformatic analysis of the cerato-platanin protein family in Dikarya. - Physiological response and sulfur metabolism of the V dahliae-infected tomato plants in tomato/potato onion companion cropping. - Partnerships between Ambrosia beetles and Fungi: lineage-specific promiscuity among vectors of the Laurel wilt pathogen, Raffaelea lauricola. - accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions
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