Genome-wide analysis of Claviceps paspali: insights into the secretome of the main species causing ergot disease in Paspalum spp

Background The phytopatogen Claviceps paspali is the causal agent of Ergot disease in Paspalum spp., which includes highly productive forage grasses such as P. dilatatum. This disease impacts dairy and beef production by affecting seed quality and producing mycotoxins that can affect performance in feeding animals. The molecular basis of pathogenicity of C. paspali remains unknown, which makes it more difficult to find solutions for this problem. Secreted proteins are related to fungi virulence and can manipulate plant immunity acting on different subcellular localizations. Therefore, identifying and characterizing secreted proteins in phytopathogenic fungi will provide a better understanding of how they overcome host defense and cause disease. The aim of this work is to analyze the whole genome sequences of three C. paspali isolates to obtain a comparative genome characterization based on possible secreted proteins and pathogenicity factors present in their genome. In planta RNA-seq analysis at an early stage of the interaction of C. paspali with P. dilatatum stigmas was also conducted in order to determine possible secreted proteins expressed in the infection process. Results C. paspali isolates had compact genomes and secretome which accounted for 4.6–4.9% of the predicted proteomes. More than 50% of the predicted secretome had no homology to known proteins. RNA-Seq revealed that three protein-coding genes predicted as secreted have mayor expression changes during 1 dpi vs 4 dpi. Also, three of the first 10 highly expressed genes in both time points were predicted as effector-like. CAZyme-like proteins were found in the predicted secretome and the most abundant family could be associated to pectine degradation. Based on this, pectine could be a main component affected by the cell wall degrading enzymes of C. paspali. Conclusions Based on predictions from DNA sequence and RNA-seq, unique probable secreted proteins and probable pathogenicity factors were identified in C. paspali isolates. This information opens new avenues in the study of the biology of this fungus and how it modulates the interaction with its host. Knowledge of the diversity of the secretome and putative pathogenicity genes should facilitate future research in disease management of Claviceps spp.

New Draft Genome Sequence of the Ergot Disease Fungus Claviceps paspali

ABSTRACT Here, we report a new draft genome sequence of an isolate of the ascomycete Claviceps paspali that is responsible for ergot disease in grasses of the Paspalum genus. This new draft genome sequence will provide useful data for evaluating intraspecies and interspecies genome variation in C. paspali and other Claviceps genus members.

Neurological diseases in cattle caused by plants and mycotoxins in Santa Catarina state, Brazil

ABSTRACT: This study described the epidemiological and clinical-pathological aspects of 25 outbreaks of neurological diseases in cattle caused by plants and mycotoxins in Santa Catarina state. Six of them were due to Sida carpinifolia poisoning, five to Solanum fastigiatum, five to Phalaris angusta, three to Claviceps paspali, three to Claviceps purpurea, and three outbreaks were of unknown etiology. The clinical signs observed in the affected cattle were mild to severe and characterized by generalized muscle tremors, incoordination, hypermetria, wide-based stance, intentional head tremors, dull staring eyes, and frequent ear twitching, with convulsions in some cases. At necropsy, lesions were observed only for P. angusta poisoning, characterized by gray-greenish discoloration in thalamus and midbrain. Microscopically, rarefaction and/or disappearance of Purkinje neurons with substitution by Bergmann cells were observed for S. carpinifolia and S. fastigiatum poisoning. For P. angusta poisoning, thin granular brown-yellowish pigment was observed in the cytoplasm of some neurons. Gross and microscopic findings were not observed in three outbreaks of tremorgenic disease of unknown etiology. Experiments conducted with leaves, flowers and seeds of Ipomoea indivisa and Ipomoea triloba, as well as with maize and soybean residues contaminated with Ipomoea spp. did not reproduced clinical signs.

The Kuwahara Synthesis of Paspalinine

The hexacyclic indole diterpenoid paspalinine 3, produced by the ergot Claviceps paspali, induces tremors in domestic animals that graze on pasture grass infected by that fungus. Shigefumi Kuwahara of Tohoku University envisioned (Angew. Chem. Int. Ed. 2012, 51, 12833) assembly of the tetracyclic nucleus of 3 by the oxidative cyclization of 1 to 2. The challenge, then, was the assembly of the angularly substituted trans–anti-trans-6-6-5 alicyclic nucleus of 1. The synthesis began with the Wieland-Miescher ketone 4, which is available commercially in enantiomerically pure form. Exposure to ethylene glycol under acidic conditions converted 4 to the known monoketal 5. The alkylation with 6 followed by selective hydrolysis gave 7 as an inconsequential mixture of diastereomers. Exposure to base effected both equilibration to the more stable equatorial diastereomer and cyclization to give the enone 8. There was still the problem of the introduction of the axial angular methyl group. This was solved by selective reduction of 8 to the endo alcohol. Hydroxyl-directed Simmons-Smith cyclopropanation followed by oxidation gave 9. The enolate resulting from the dissolving metal reduction of 9 was trapped with Comins reagent to generate the enol triflate 10. Acetanilide 11 was converted to the stannane 12 by ortho metalation following the literature protocol. Pd-mediated coupling of 12 with 10 proceeded efficiently to give 1. Oxidation with stoichiometric Pd trifluoroacetate then induced Type 5 indole formation (Tetrahedron 2011, 67, 7195). Hydrolysis and conjugation completed the synthesis of 2. To introduce the sidechain, the enone was converted to the allyl enol carbonate. Pd-mediated rearrangement converted that to the α-allyl enone, which participated in Ru-catalyzed metathesis with 14 to give 15. Asymmetric dihydroxylation proceeded with only modest diastereoselectivity, but only the desired diastereomer cyclized onto the ketone to give 16. The ketone 16 was an intermediate in Smith’s earlier synthesis. Professor Kuwahara devised an alternate end game, selenylation followed by oxidation and sigmatropic rearrangement, to install the axial OH and complete the synthesis of paspalinine 3.