Genetic Mapping, Candidate Gene Identification and Marker Validation for Host Plant Resistance to the Race 4 of Fusarium oxysporum f. sp. cubense Using Musa acuminata ssp. malaccensis.

Fusarium wilt of banana is a devastating disease that has decimated banana production worldwide. Host resistance to Fusarium oxysporum f. sp. Cubense (Foc), the causal agent of this disease, is genetically dissected in this study using two Musa acuminata ssp. Malaccensis segregating populations, segregating for Foc Tropical (TR4) and Subtropical (STR4) race 4 resistance. Marker loci and trait association using 11 SNP-based PCR markers allowed the candidate region to be delimited to a 12.9 cM genetic interval corresponding to a 959 kb region on chromosome 3 of 'DH-Pahang' reference assembly v4. Within this region, there was a cluster of pattern recognition receptors, namely leucine-rich repeat ectodomain containing receptor-like protein kinases, cysteine-rich cell-wall-associated protein kinases, and leaf rust 10 disease-resistance locus receptor-like proteins, positioned in an interspersed arrangement. Their transcript levels were rapidly upregulated in the resistant progenies but not in the susceptible F2 progenies at the onset of infection. This suggests that one or several of these genes may control resistance at this locus. To confirm the segregation of single-gene resistance, we generated an inter-cross between the resistant parent 'Ma850' and a susceptible line 'Ma848', to show that the STR4 resistance co-segregated with marker '28820' at this locus. Finally, an informative SNP marker 29730 allowed the locus-specific resistance to be assessed in a collection of diploid and polyploid banana plants. Of the 60 lines screened, 22 lines were predicted to carry resistance at this locus, including lines known to be TR4-resistant, such as 'Pahang', 'SH-3362', 'SH-3217', 'Ma-ITC0250', and 'DH-Pahang/CIRAD 930'. Additional screening in the International Institute for Tropical Agriculture's collection suggests that the dominant allele is common among the elite 'Matooke' NARITA hybrids, as well as in other triploid or tetraploid hybrids derived from East African highland bananas. Fine mapping and candidate gene identification will allow characterization of molecular mechanisms underlying the TR4 resistance. The markers developed in this study can now aid the marker-assisted selection of TR4 resistance in breeding programs around the world.

A Polyphasic Approach Reveals Novel Genotypes and Updates the Genetic Structure of the Banana Fusarium Wilt Pathogen.

Fusarium oxysporum f. sp. cubense (Foc) is a soil-borne fungus that causes Fusarium wilt, a destructive plant disease that has resulted in devastating economic losses to banana production worldwide. The fungus has a complex evolutionary history and taxonomic repute and consists of three pathogenic races and at least 24 vegetative compatibility groups (VCGs). Surveys conducted in Asia, Africa, the Sultanate of Oman and Mauritius encountered isolates of F. oxysporum pathogenic to banana that were not compatible to any of the known Foc VCGs. Genetic relatedness between the undescribed and known Foc VCGs were determined using a multi-gene phylogeny and diversity array technology (DArT) sequencing. The presence of putative effector genes, the secreted in xylem (SIX) genes, were also determined. Fourteen novel Foc VCGs and 17 single-member VCGs were identified. The multi-gene tree was congruent with the DArT-seq phylogeny and divided the novel VCGs into three clades. Clustering analysis of the DArT-seq data supported the separation of Foc isolates into eight distinct clusters, with the suite of SIX genes mostly conserved within these clusters. Results from this study indicates that Foc is more diverse than hitherto assumed.

The M35 Metalloprotease Effector FocM35_1 Is Required for Full Virulence of Fusarium oxysporum f. sp. cubense Tropical Race 4.

Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) causes Fusarium wilt of banana, the most devastating disease on a banana plant. The genome of Foc TR4 encodes many candidate effector proteins. However, little is known about the functions of these effector proteins on their contributions to disease development and Foc TR4 virulence. Here, we discovered a secreted metalloprotease, FocM35_1, which is an essential virulence effector of Foc TR4. FocM35_1 was highly upregulated during the early stages of Foc TR4 infection progress in bananas. The FocM35_1 knockout mutant compromised the virulence of Foc TR4. FocM35_1 could interact with the banana chitinase MaChiA, and it decreased banana chitinase activity. FocM35_1 induced cell death in Nicotiana benthamiana while suppressing the INF1-induced hypersensitive response (HR), and its predicted enzymatic site was required for lesion formation and the suppression to INF1-induced HR on N. benthamiana leaves. Importantly, treatment of banana leaves with recombinant FocM35_1 accelerates Foc TR4 infection. Collectively, our study provides evidence that metalloprotease effector FocM35 seems to contribute to pathogen virulence by inhibiting the host immunity.