Requirement of Two Acyltransferases for 4-O-Acylation during Biosynthesis of Harzianum A, an Antifungal Trichothecene Produced by Trichoderma arundinaceum

Trichothecenes are sesquiterpenoid toxins produced by multiple fungi, including plant pathogens, entomopathogens, and saprotrophs. Most of these fungi have the acyltransferase-encoding gene tri18. Even though its function has not been determined, tri18 is predicted to be involved in trichothecene biosynthesis because of its pattern of expression and its location near other trichothecene biosynthetic genes. Here, molecular genetic, precursor feeding, and analytical chemistry experiments indicate that in the saprotroph Trichoderma arundinaceum the tri18-encoded acyltransferase (TRI18) and a previously characterized acyltransferase (TRI3) are required for conversion of the trichothecene biosynthetic intermediate trichodermol to harzianum A, an antifungal trichothecene analog with an octa-2,4,6-trienedioyl acyl group. On the basis of the results, we propose that TRI3 catalyzes trichothecene 4-O-acetylation, and subsequently, TRI18 catalyzes replacement of the resulting acetyl group with octa-2,4,6-trienedioyl to form harzianum A. Thus, the findings provide evidence for a previously unrecognized two-step acylation process during trichothecene biosynthesis in T. arundinaceum and possibly other fungiSIThe Spanish Ministry of Economy and Competitiveness supported this work (MINECO-AGL2015-70671-C2-2-R to S.G.), and the University of León granted L.L. a fellowshi

Botrydial and botcinins produced by Botrytis cinerea regulate the expression of Trichoderma arundinaceum genes involved in trichothecene biosynthesis

Trichoderma arundinaceum IBT 40837 (Ta37) and Botrytis cinerea produce the sesquiterpenes harzianum A (HA) and botrydial (BOT), respectively, and also the polyketides aspinolides and botcinins (Botcs), respectively. We analysed the role of BOT and Botcs in the Ta37-B. cinerea interaction, including the transcriptomic changes in the genes involved in HA (tri) and ergosterol biosynthesis, as well as changes in the level of HA and squalene-ergosterol. We found that, when confronted with B. cinerea, the tri biosynthetic genes were up-regulated in all dual cultures analysed, but at higher levels when Ta37 was confronted with the BOT non-producer mutant bcbot2Δ. The production of HA was also higher in the interaction area with this mutant. In Ta37-bcbot2Δ confrontation experiments, the expression of the hmgR gene, encoding the 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is the first enzyme of the terpene biosynthetic pathway, was also up-regulated, resulting in an increase in squalene production compared with the confrontation with B. cinerea B05.10. Botcs had an up-regulatory effect on the tri biosynthetic genes, with BotcA having a stronger effect than BotcB. The results indicate that the interaction between Ta37 and B. cinerea exerts a stimulatory effect on the expression of the tri biosynthetic genes, which, in the interaction zone, can be attenuated by BOT produced by B. cinerea B05.10. The present work provides evidence for a metabolic dialogue between T. arundinaceum and B. cinerea that is mediated by sesquiterpenes and polyketides, and that affects the outcome of the interaction of these fungi with each other and their environmentSIFunding was obtained from the Junta de Castilla y Leon (SA260A11-2,LE125A12-2 and LE228U14) and Spanish Government grants MICINN-AGL2009-13431-C02-02, MINECO-AGL2012-40041-C02-01, AGL2012-40041-C02-02 and AGL2012-39798-C02-01. MGM and II-B were granted fellowships from the Spanish Ministry of Science and Innovation(AP2007-02835, BES-2013-063411

Effect of Farnesol in Trichoderma Physiology and in Fungal–Plant Interaction

[EN] Farnesol is an isoprenoid intermediate in the mevalonate (MVA) pathway and is produced by the dephosphorylation of farnesyl diphosphate. Farnesol plays a central role in cell growth and differentiation, controls production of ubiquinone and ergosterol, and participates in the regulation of filamentation and biofilm formation. Despite these important functions, studies of farnesol in filamentous fungi are limited, and information on its effects on antifungal and/or biocontrol activity is scarce. In the present article, we identified the Trichoderma harzianum gene dpp1, encoding a diacylglycerol pyrophosphatase that catalyzes production of farnesol from farnesol diphosphate. We analyzed the function of dpp1 to address the importance of farnesol in Trichoderma physiology and ecology. Overexpression of dpp1 in T. harzianum caused an expected increase in farnesol production as well as a marked change in squalene and ergosterol levels, but overexpression did not affect antifungal activity. In interaction with plants, a dpp1-overexpressing transformant acted as a sensitizing agent in that it up-regulated expression of plant defense salicylate-related genes in the presence of a fungal plant pathogen. In addition, toxicity of farnesol on Trichoderma and plants was examined. Finally, a phylogenetic study of dpp1 was performed to understand its evolutionary history as a primary metabolite gene. This article represents a step forward in the acquisition of knowledge on the role of farnesol in fungal physiology and in fungus-environment interactionsSIThis research was funded by the Spanish I+D+i Grants AGL2012-40041-C02-02, AGL2015-70671-C2-2-R, RTI2018-099600-B-I00 and PID2021-123874OB-I00, financed by the MCIN/ AEI/10.13039/501100011033. GC-H was awarded with a Grant from the Ministry of Education, Culture, and Sport (Spain) (Grant number FPU15/04681). NM-R was awarded with a Grant from the Junta de Castilla y León (Spain) (ORDEN EDU/875/2021, 13 July 2021

Evolution of structural diversity of trichothecenes, a family of toxins produced by plant pathogenic and entomopathogenic fungi

[EN] Trichothecenes are a family of terpenoid toxins produced by multiple genera of fungi, including plant and insect pathogens. Some trichothecenes produced by the fungus Fusarium are among the mycotoxins of greatest concern to food and feed safety because of their toxicity and frequent occurrence in cereal crops, and trichothecene production contributes to pathogenesis of some Fusarium species on plants. Collectively, fungi produce over 150 trichothecene analogs: i.e., molecules that share the same core structure but differ in patterns of substituents attached to the core structure. Here, we carried out genomic, phylogenetic, gene-function, and analytical chemistry studies of strains from nine fungal genera to identify genetic variation responsible for trichothecene structural diversity and to gain insight into evolutionary processes that have contributed to the variation. The results indicate that structural diversity has resulted from gain, loss, and functional changes of trichothecene biosynthetic (TRI) genes. The results also indicate that the presence of some substituents has arisen independently in different fungi by gain of different genes with the same function. Variation in TRI gene duplication and number of TRI loci was also observed among the fungi examined, but there was no evidence that such genetic differences have contributed to trichothecene structural variation. We also inferred ancestral states of the TRI cluster and trichothecene biosynthetic pathway, and proposed scenarios for changes in trichothecene structures during divergence of TRI cluster homologs. Together, our findings provide insight into evolutionary processes responsible for structural diversification of toxins produced by pathogenic fungiSISG received funding from the Spanish Ministry of Economy and Competitiveness (grant number MINECO-AGL2015-70671-C2-2-R). TL received funding from the National Institute of Agricultural Sciences, Rural Development Administration (grant number PJ00843203). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Effects of trichothecene production by Trichoderma arundinaceum isolates from bean-field soils on the defense response, growth and development of bean plants (Phaseolus vulgaris)

[EN] The trichothecene toxin-producing fungus Trichoderma arundinaceum has potential as a biological control agent. However, most biocontrol studies have focused only on one strain, IBT 40837. In the current study, three Trichoderma isolates recovered from bean-field soils produced the trichothecene harzianum A (HA) and trichodermol, the latter being an intermediate in the HA biosynthesis. Based on phylogenetic analysis, the three isolates were assigned to the species T. arundinaceum. Their genome sequences had a high degree of similarity to the reference IBT 40837 strain, in terms of total genome size, number of predicted genes, and diversity of putative secondary metabolite biosynthetic gene clusters. HA production by these bean-field isolates conferred significant in vitro antifungal activity against Rhizoctonia solani and Sclerotinia sclerotiorum, which are some of the most important bean pathogens. Furthermore, the bean-field isolates stimulated germination of bean seeds and subsequent growth of above ground parts of the bean plant. Transcriptomic analysis of bean plants inoculated with these T. arundinaceum bean-field soil isolates indicated that HA production significantly affected expression of plant defense-related genes; this effect was particularly significant in the expression of chitinase-encoding genes. Together, these results indicate that Trichoderma species producing non-phytotoxic trichothecenes can induce defenses in plants without negatively affecting germination and developmentSIThis work is a part of the Spanish I+D+i Grants RTI2018-099600-B-I00 and PID2021-123874OB-I00, financed by the MCIN/ AEI/10.13039/501100011033. GC-H was awarded with a Grant from the Ministry of Education, Culture, and Sport (Spain) (Grant number FPU15/04681). NM-R was awarded with a Grant from the Junta de Castilla y León (Spain) (ORDEN EDU/875/2021, July 13th, 2021

Distribution, Function, and Evolution of a Gene Essential for Trichothecene Toxin Biosynthesis in Trichoderma

[EN] Trichothecenes are terpenoid toxins produced by species in 10 fungal genera, including species of Trichoderma. The trichothecene biosynthetic gene (tri) cluster typically includes the tri5 gene, which encodes a terpene synthase that catalyzes formation of trichodiene, the parent compound of all trichothecenes. The two Trichoderma species, Trichoderma arundinaceum and T. brevicompactum, that have been examined are unique in that tri5 is located outside the tri cluster in a genomic region that does not include other known tri genes. In the current study, analysis of 35 species representing a wide range of the phylogenetic diversity of Trichoderma revealed that 22 species had tri5, but only 13 species had both tri5 and the tri cluster. tri5 was not located in the cluster in any species. Using complementation analysis of a T. arundinaceum tri5 deletion mutant, we demonstrated that some tri5 homologs from species that lack a tri cluster are functional, but others are not. Phylogenetic analyses suggest that Trichoderma tri5 was under positive selection following its divergence from homologs in other fungi but before Trichoderma species began diverging from one another. We propose two models to explain these diverse observations. One model proposes that the location of tri5 outside the tri cluster resulted from loss of tri5 from the cluster in an ancestral species followed by reacquisition via horizontal transfer. The other model proposes that in species that have a functional tri5 but lack the tri cluster, trichodiene production provides a competitive advantage.S

Franchises lost and gained: post-coloniality and the development of women’s rights in Canada

The Canadian constitution is to some extent characterised by its focus on equality, and in particular gender equality. This development of women’s rights in Canada and the greater engagement of women as political actors is often presented as a steady linear process, moving forwards from post-enlightenment modernity. This article seeks to disturb this ‘discourse of the continuous,’ by using an analysis of the pre-confederation history of suffrage in Canada to both refute a simplistic linear view of women’s rights development and to argue for recognition of the Indigenous contribution to the history of women’s rights in Canada. The gain of franchise and suffrage movements in Canada in the late nineteenth and early twentieth century are, rightly, the focus of considerable study (Pauker 2015), This article takes an alternative perspective. Instead, it examines the exercise of earlier franchises in pre-confederation Canada. In particular it analyses why franchise was exercised more widely in Lower Canada and relates this to the context of the removal of franchises from women prior to confederation

Global economic burden of unmet surgical need for appendicitis

Background: There is a substantial gap in provision of adequate surgical care in many low-and middle-income countries. This study aimed to identify the economic burden of unmet surgical need for the common condition of appendicitis. Methods: Data on the incidence of appendicitis from 170 countries and two different approaches were used to estimate numbers of patients who do not receive surgery: as a fixed proportion of the total unmet surgical need per country (approach 1); and based on country income status (approach 2). Indirect costs with current levels of access and local quality, and those if quality were at the standards of high-income countries, were estimated. A human capital approach was applied, focusing on the economic burden resulting from premature death and absenteeism. Results: Excess mortality was 4185 per 100 000 cases of appendicitis using approach 1 and 3448 per 100 000 using approach 2. The economic burden of continuing current levels of access and local quality was US $92 492 million using approach 1 and$73 141 million using approach 2. The economic burden of not providing surgical care to the standards of high-income countries was $95 004 million using approach 1 and$75 666 million using approach 2. The largest share of these costs resulted from premature death (97.7 per cent) and lack of access (97.0 per cent) in contrast to lack of quality. Conclusion: For a comparatively non-complex emergency condition such as appendicitis, increasing access to care should be prioritized. Although improving quality of care should not be neglected, increasing provision of care at current standards could reduce societal costs substantially

Pooled analysis of WHO Surgical Safety Checklist use and mortality after emergency laparotomy

Background The World Health Organization (WHO) Surgical Safety Checklist has fostered safe practice for 10 years, yet its place in emergency surgery has not been assessed on a global scale. The aim of this study was to evaluate reported checklist use in emergency settings and examine the relationship with perioperative mortality in patients who had emergency laparotomy. Methods In two multinational cohort studies, adults undergoing emergency laparotomy were compared with those having elective gastrointestinal surgery. Relationships between reported checklist use and mortality were determined using multivariable logistic regression and bootstrapped simulation. Results Of 12 296 patients included from 76 countries, 4843 underwent emergency laparotomy. After adjusting for patient and disease factors, checklist use before emergency laparotomy was more common in countries with a high Human Development Index (HDI) (2455 of 2741, 89.6 per cent) compared with that in countries with a middle (753 of 1242, 60.6 per cent; odds ratio (OR) 0.17, 95 per cent c.i. 0.14 to 0.21, P <0001) or low (363 of 860, 422 per cent; OR 008, 007 to 010, P <0.001) HDI. Checklist use was less common in elective surgery than for emergency laparotomy in high-HDI countries (risk difference -94 (95 per cent c.i. -11.9 to -6.9) per cent; P <0001), but the relationship was reversed in low-HDI countries (+121 (+7.0 to +173) per cent; P <0001). In multivariable models, checklist use was associated with a lower 30-day perioperative mortality (OR 0.60, 0.50 to 073; P <0.001). The greatest absolute benefit was seen for emergency surgery in low- and middle-HDI countries. Conclusion Checklist use in emergency laparotomy was associated with a significantly lower perioperative mortality rate. Checklist use in low-HDI countries was half that in high-HDI countries.Peer reviewe

Global variation in anastomosis and end colostomy formation following left-sided colorectal resection

Background End colostomy rates following colorectal resection vary across institutions in high-income settings, being influenced by patient, disease, surgeon and system factors. This study aimed to assess global variation in end colostomy rates after left-sided colorectal resection. Methods This study comprised an analysis of GlobalSurg-1 and -2 international, prospective, observational cohort studies (2014, 2016), including consecutive adult patients undergoing elective or emergency left-sided colorectal resection within discrete 2-week windows. Countries were grouped into high-, middle- and low-income tertiles according to the United Nations Human Development Index (HDI). Factors associated with colostomy formation versus primary anastomosis were explored using a multilevel, multivariable logistic regression model. Results In total, 1635 patients from 242 hospitals in 57 countries undergoing left-sided colorectal resection were included: 113 (6·9 per cent) from low-HDI, 254 (15·5 per cent) from middle-HDI and 1268 (77·6 per cent) from high-HDI countries. There was a higher proportion of patients with perforated disease (57·5, 40·9 and 35·4 per cent; P < 0·001) and subsequent use of end colostomy (52·2, 24·8 and 18·9 per cent; P < 0·001) in low- compared with middle- and high-HDI settings. The association with colostomy use in low-HDI settings persisted (odds ratio (OR) 3·20, 95 per cent c.i. 1·35 to 7·57; P = 0·008) after risk adjustment for malignant disease (OR 2·34, 1·65 to 3·32; P < 0·001), emergency surgery (OR 4·08, 2·73 to 6·10; P < 0·001), time to operation at least 48 h (OR 1·99, 1·28 to 3·09; P = 0·002) and disease perforation (OR 4·00, 2·81 to 5·69; P < 0·001). Conclusion Global differences existed in the proportion of patients receiving end stomas after left-sided colorectal resection based on income, which went beyond case mix alone