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Horizontal gene transfer of Fhb7 for Fusarium head blight resistance in wheat

F. graminearum is the prominent pathogen of wheat FHB in China, USA, Canada, Europe and many other countries, which threat for global wheat production and food safety. Wheat relatives have proven to be alternative sources for improvement of resistance to both biotic and abiotic stresses in wheat. Th. elongatum has been used into wheat for resistance breeding because of its potential of FHB resistance and strong growth ability. Fhb7 plays a main role in FHB resistance in Th. Elongatum, However, the E reference genome of Th. Elongatum is not available and molecular identity and mechanisms of FHB resistance remains equivocal.

In this study, high-quality diploid Th. Elongatum genome was assembled by combining Illumina, PacBio, Bionano and Hi-C technologies. The assembly was validated using independent BAC sequences, genetic maps of related species and commonly used software programs. Genes, repetitive DNA, and other genomic features in the assembly were annotated to reveal the landscape of the species, and relationship with wheat and other related species by in-depth comparative analyses among these species. Genetic markers in the Fhb7 region were developed using the reference genome sequence, and used to screen recombinants for fine mapping to identify Fhb7 candidate gene. The candidate gene was functionally validated by virus-induced gene silencing (VIGS), EMS-induced mutation, and transgenic approaches. FHB resistance was evaluated by inoculation of Fusarium conidial suspensions on wheat spikes, leaves or crown. The LC-HRMS(/MS) analysis was employed to infer the biochemical structure of trichothecene-glutathione adducts catalyzed by Fhb7. Fhb7 was introgressed into diverse wheat backgrounds using distant hybridization and conventional breeding, and the presence of alien chromatin in wheat was validated by genomic in situ hybridization (GISH).

1. Th. elongatum genome assembly and evolution
A total of 141 scaffolds were anchored and oriented onto 7 pseudo-chromosomes, which account for 95% of the estimated genome size (4.78 Gb). Results shows a 4.63 Gb assembly with a contig N50 size of 2.15 Mb and a scaffold N50 size of 73.24 Mb. A total of 44,474 high-confidence protein-coding genes were predicted, with about 81.29% of the Th. elongatum assembly being annotated as repetitive elements. Gene family analysis of E, A, B, D and other Triticeae genomes indicates a branching time for Th. elongatum and Triticum of about 4.77-4.96 Mya when a nucleotide substitution rate of 6.5*10-9 was used. The three wheat sub-genomes are more closely related to the E genome of Th. elongatum than they are to the R genome of rye, another frequently employed species used in wheat distant hybridization (Fig. 1A). Substantial co-linearity notwithstanding, 18 fragmental inversions were identified between the E genome and the wheat sub-genomes, with size ranging from 1.5 to 18 Mb.

2. Fine mapping of Fhb7
An apparent RGA expansion on the distal end of the long arm of chromosome 7E,indicating Fhb7 is posited on 7E. Researchers further confirmed that Fhb7 is positioned between the XSdauK79 and XSdauK80 markers, within a ~1.2Mb region through a cross screening between FHB-susceptible substitution line and an FHB- resistant substitution line. Analysis of the RNA-seq data of E reference genome from Th. elongatum spikes identified 8 expressed genes in the Fhb7 region. However, only two candidate genes (Tel7E01T1020600.1 and Tel7E01T1021800.1) were expressed in a manner specific. BAC sequencing and analysis of phenotypic data verified that Fhb7 is located between the XsdauK86 and XsdauK88 markers, thereby delineating this locus to a 245 kb region containing a single expressed gene: Tel7E01T1020600.1(Fig. 1C). To confirm Tel7E01T1020600.1 as Fhb7, a construct with the native promoter and the 846 bp coding sequence of this gene was introduced into the FHB-disease-susceptible wheat cultivar ‘KN199’ and assessed three independent T3 transgenic plants. The Fusarium-inoculated transgenic plants exhibited significantly lower FHB with dramatically fewer diseased spikelets per spike than the control.

3. Origin of Fhb7
A BLAST search of the Fhb7 sequence against the NCBI GenBank database did not find any homolog of Fhb7 in not only the Triticum genus, also the entire plant kingdom. A phylogenetic analysis of the Fhb7 sequence revealed its distribution among Epichloë species, endophytic fungi of temperate grasses. Thus, the occurrence of the Fhb7 gene in the Th. elongatum genome might be due to fungus-to-plant horizontal gene transfer (FP-HGT) event. This FP-HGT event apparently occurred after the divergence of the E genome from Triticum sp. but before the formation of the decaploid Th. Ponticum (Fig. 2A). Based on sequence similarity, the sequence was transferred into the diploid E genome as a short fragment, including the 846 bp coding sequence for Fhb7, a 32 bp sequence before the start codon, and a 19 bp sequence after the stop codon. At the position 535 bp upstream of Fhb7’s start codon in the E genome, another 90 bp sequence shows high identity to a sequence in E. aotearoae, suggesting the possibility that a larger sequence was initially transferred to Th. elongatum, but late mutations occurred in the transferred sequence.

4. FHB resistance mechanism of Fhb7
Phylogenetic analysis of the GST superfamily showed that Fhb7 belongs to the fungal GTE (glutathione transferase etherase-related) subfamily, wherein all members contain a LigE domain, but none of which have been functionally characterized to date. Research in plant pathology about the infection progression of F. graminearum in wheat has established that the fungus starts to produce its deoxynivalenol (DON) mycotoxin—a inhibitor of protein synthesis that targets ribosomal machinery—by this 48h infection time point. Researchers therefore conducted DON assays on wheat seedlings of 7E2/7D substitution line. The result showed that the expression of Fhb7 can be induced within 6 h after DON treatment, suggesting that this putative GST enzyme may have a role in xenobiotic detoxification. To test this hypothesis, they conducted a growth inhibition assay by growing Fhb7 near-isogenic lines (NILs) and Fhb7 transgenic wheat seedlings in media containing DON, and found that the plants with Fhb7 obviously grew better (assessed as seedling length) than the plants without Fhb7 (Fig. 2C). Fhb7 confers GST activity to form a glutathione adduct of DON (DON-235 GSH) (Fig. 2D). The GSH group added by Fhb7 is attached to the C13 carbon, which disrupts the epoxy group known to be critical in DON’s toxicity.

5. Application of Fhb7 in Fusarium resistance breeding
Considering Fhb7’s functionality—enzymatic conversion of trichothecenes—researchers speculated that incorporating the Fhb7 locus into wheat may confer resistance in different genetic backgrounds without affecting yield traits. Indeed, the translocation of a short fragment (with ~16% of the 7E 269 long arm) on wheat 7D resulted in wheat lines with broad resistance to both FHB and crown rot (Fig. 3A-C). Detailed characterization of NILs (‘LX99’ background) in field conditions showed no significant difference in agronomic yield traits (e.g., thousand grain weight, flag leaf length, etc.) (Fig. 3D-E). Obvious yield penalty due to Fhb7 resistance was also not detected when it was transferred into seven additional genetic backgrounds (Fig. 3F). These results demonstrated the advantages of Fhb7-mediated resistance over other QTLs: high resistance to both FHB and crown rot, and detoxifying DON without yield penalty, thus highlighted potential utility of the Fhb7 locus in future wheat breeding for improved FHB resistance and good yield traits.

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