Cereals endophytes : biostimulation and biocontrol

Team SYMUNITY / Pascal Ratet

 

Small-grain cereals (bread and durum wheat, barley, ...) are agronomically important plant species. Like all plant crops, they are subject to abiotic or biotic attacks. In order to adapt to these different stresses, cereals set up defence and/or adaptation mechanisms. They can also interact with beneficial microorganisms enabling them to grow better (biostimulation) and/or defend themselves against pathogens (biocontrol).


The lines of research that our team is developing mainly use two cereal species: common wheat (Triticum aestivum) and Brachypodium distachyon. Various cereal-fungal pathogen interactions are also used, with a particular focus on Fusarium head blight (FHB), the main causal agent of which being the ascomycete fungus Fusarium graminearum. This fungal species is capable, during spike infection of its host (see Figure below), of producing mycotoxins, which are harmful to humans and animals, one of the most important of which is deoxynivalenol (DON).


Figure 5. Typical Fusarium Head Blight 14 days after inoculation of spikes of Brachypodium distachyon (left) and of common wheat (right). Bars: 1cm.

Two main areas are developed in the "Cereals" part of the SYMUNITY team:

  • Functional characterisation of candidate genes potentially involved in the defence against fusarium head blight
  • Studying the impact of beneficial soil microorganisms on growth and disease protection

 

 

Mechanisms of defence against fusarium head blight

 


Relationship between detoxification of DON by the plant and disease resistance


At the start of this project, few studies linking DON detoxification and resistance to Fusarium head blight had been conducted. All used heterologous systems (yeast, Arabidopsis or recombinant protein production in Escherichia coli). The work of our team consisted in testing the impact of DON glucosylation in the host system for F. graminearum by (i) functional characterisation of a UDP-glucosyltransferase (UGT) of the model cereal Brachypodium distachyon, (ii) identification and characterisation of a UGT in common wheat.


Figure 6. The Brachypodium distachyon gene Bradi5g03300 encodes a UDP-glucosyltransferase involved in the control of the colonisation of ears by Fusarium graminearum. A: Typical symptoms of fusarium disease observed 7 days after point inoculation by F. graminearum: Mock, negative control; Bd21-3, wild line; 8637-12 and 6829-7, mutant lines (TILLING); OE-9R5, 24R27, 10R14, transgenic lines suprepressing the Bradi5g03300 gene. B, Quantification of Fusarium symptoms using a 7 (grey histograms) and 14 (black histograms) days rating scale after point inoculation (n>30, error bars represent standard deviations, different letters indicate significant differences between lines; Newman and Keuls test, p-value ≤ 0.001).

Using a phylogenetic approach and transcriptomic analyses, B. distachyon genes coding for transcriptionally induced mycotoxin-induced UGTs and F. graminearum infection were identified (Schweiger et al. 2013). Among these, the Bradi5g03300 gene confers tolerance to DON in the yeast system (Schweiger et al. 2013). Functional characterisation of this gene in planta showed that the Bradi5g03300 UGT conjugates DON to DON-3-O¬-glucose in planta and that its overexpression allows the establishment of resistance not only to the colonisation of the ears but also to primary infection by F. graminearum (Pasquet et al. 2016, see Figre above). On the other hand, the expression of the B. distachyon gene in a spring wheat variety, while allowing the conjugation of DON in planta, only showed an effect on ear colonisation (type II resistance) (Gatti et al. 2019).


A synteny approach between Brachypodium and common wheat (GDEC collaboration, Clermont-Ferrand, France) has identified potential orthologs of the Bradi5g03300 gene in common wheat. One of them, which expression is strongly induced under conditions of infection by F. graminearum, was functionally characterized by genetic transformation of B. distachyon. We were able to show that the wheat UGT was capable of conferring type II resistance to Fusarium head blight significantly reducing the level of mycotoxins in planta (Gatti et al. 2018).

 

Plant genes as interesting candidates involved in FHB resistance or defense

 

Transcriptomics analyses were performed on B. distachyon plants either infected with DON-producing or DON non producing strains of F. graminearum or inoculated with DON itselfin order to identify candidate genes potentially involved in detoxification as well as metabolic pathways deregulated by the mycotoxin. A number of differentially expressed genes were retrieved, some of them encoding protein functions known to be involved in detoxification processes such as UGTs, CYPs (cytochrome P450) and MATEs(multidrug and toxic compound extrusion) (Pasquet et al. 2014). Among these candidates, we favoured a gene encoding a CYP (CYP711A29). Such enzymes are usually occurring in the early steps of detoxification processes in plants, allowing a chemical modification of metabolites which are then more amenable to conjugation performed either by UGTs or GSTs. DON exhibiting a number of hydroxyl groups can readily be conjugated. Our hypothesis was that CYP711A29 is able to modify the DON glucoside into an even less toxic molecule. This hypothesis has been tested in the frame of V. Changenet’s PhD work (2015-2018) following a similar strategy as the one used to study Bradi5g03300 (construction of B. distachyon lines over-expressing the gene; search for mutant lines in the TILLING collection; functional characterization of these lines and in vitro characterization of CYP711A29 activity and determination of the end product by LC-MS-MS (collaboration with D. Werck, IBMP, Strasbourg). These analyses have shown that the B. distachyon CYP711A29 is involved in orobanchol biosynthesis (Changenet et al., submitted). Experiments are underway to determine whether strigolactones are part of the plant defense reactions or if the fungal pathogen diverts this biosynthetic pathway to favour its establishment in the plant tissues.

 

 

Impact of beneficial microorganisms on cereal growth and resistance to pathogens

 

Recently, the team has developed a research theme aimed at studying the impact of beneficial microorganisms in dublé nutrition/growth or in its interactions with phytopathogenic fungi.


Interactions between cereals and diazotrophic bacteria


Field crops such as wheat, barley and maize require the addition of chemical fertilisers for their growth. We would like to know if these crops are capable of interacting with micro-organisms fixing atmospheric nitrogen. Nitrogen-fixing endophytes (N2) are, for example, present in sugar cane. We have isolated root endophytes capable of fixing nitrogen in vitro from wheat grown organically (field plants grown without chemical fertiliser). Several bacteria were isolated and the genome of one of them was sequenced (unpublished data). Preliminary experiments indicate a close association of one of these strains with wheat roots. Further experiments will show whether this bacterium can behave as an endophyte.

 


Trichoderma sp: biocontrol agent, biostimulating agent or both?


A second part is studying the impact of interaction with a strain of Trichoderma sp. on : (1) stimulation of plant growth and (2) protection against fusarium head blight depending on the mode of application. This collaborative project (IJPB Versailles, IPS2, Plant2Pro) will provide a better understanding of the mechanisms involved and their potential interaction(s).


Based on the same strain of Trichoderma sp. a biocontrol solution against Fusarium head blight is currently being evaluated in the field (SATT Paris-Saclay, see Figure below).


Figure 7. FHB symptoms on wheat spikes 14 days after inoculation with Fusarium graminearum spores with (+ Biocontrol agent, right) or without (Control, left) pretreatment with spores of Trichoderma sp.