Surface-Imprinted Polysiloxane with Recognition Ability Based on an ITO Layer for Rapid Detection of Fusarium oxysporum f. sp. cubense by the Naked Eye.

Direct observation by the naked eye of fluorescence-stained microbes adsorbed on surface imprinted polymers (SIPs) is highly challenging and limited by speed, accuracy and the semiquantitative nature of the method. In this study, we tested for the presence of spores of Fusarium oxysporum f. sp. cubense race 4 (Foc4), which cause severe banana Fusarium wilt disease and reduces the area of banana plants. This kind of spore can become dormant in soil, which means that the detection of secreted molecules (molecular imprinting) in soil may be inaccurate; detection methods such as polymerase chain reaction (PCR) and Raman spectroscopy are more accurate but time-consuming and inconvenient. Therefore, a semiquantitative and rapid SIP detection method for Foc4 was proposed. Based on the ITO conductive layer, a reusable and naked-eye-detectable Foc4-PDMS SIP film was prepared with a site density of approximately 9000 mm-2. Adsorption experiments showed that when the Foc4 spore concentration was between 104 to 107 CFU/mL, the number of Foc4 spores adsorbed and the fluorescence intensity were strongly correlated with the concentration and could be fully distinguished by the naked eye after fluorescence staining. Adsorption tests on other microbes showed that the SIP film completely recognized only the Foc series. All the results were highly consistent with the naked-eye observations after fluorescence staining, and the results of the Foc4-infected soil experiment were also close to the ideal situation. Taken together, these results showed that Foc4-PDMS SIPs have the ability to rapidly and semiquantitatively detect the concentration of Foc in soil, which can provide good support for banana cultivation. This method also has potential applications in the detection of other fungal diseases.

Secreted in Xylem (SIX) genes in Fusarium oxysporum f.sp. cubense (Foc) unravels the potential biomarkers for early detection of Fusarium wilt disease.

Secreted in Xylem (SIX) are small effector proteins released by Fusarium oxysporum f.sp. cubense (Foc) into the plant's xylem sap disrupting the host's defence responses causing Fusarium wilt disease resulting in a significant decline in banana crop yields and economic losses. Notably, different races of Foc possess unique sets of SIX genes responsible for their virulence, however, these genes remain underutilized, despite their potential as biomarkers for early disease detection. Herein, we identified seven SIX genes i.e. SIX1, SIX2, SIX4, SIX6, SIX8a, SIX9a and SIX13 present in Foc Tropical Race 4 (FocTR4), while only SIX9b in Foc Race 1 (Foc1). Analysis of SIX gene expression in infected banana roots revealed differential patterns during infection providing valuable insights into host-pathogen interactions, virulence level, and early detection time points. Additionally, a comprehensive analysis of virulent Foc1_C2HIR and FocTR4_C1HIR isolates yielded informative genomic insights. Hence, these discoveries contribute to our comprehension of potential disease control targets in these plants, as well as enhancing plant diagnostics and breeding programs.

Biodiversity of Trichoderma species of healthy and Fusarium wilt-infected banana rhizosphere soils in Tenerife (Canary Islands, Spain).

Banana (Musa acuminata) is the most important crop in the Canary Islands (38.9% of the total cultivated area). The main pathogen affecting this crop is the soil fungal Fusarium oxysporum f. sp. cubense subtropical race 4 (Foc-STR4), for which there is no effective control method under field conditions. Therefore, the use of native biological control agents may be an effective and sustainable alternative. This study aims to: (i) investigate the diversity and distribution of Trichoderma species in the rhizosphere of different banana agroecosystems affected by Foc-STR4 in Tenerife (the island with the greatest bioclimatic diversity and cultivated area), (ii) develop and preserve a culture collection of native Trichoderma species, and (iii) evaluate the influence of soil chemical properties on the Trichoderma community. A total of 131 Trichoderma isolates were obtained from 84 soil samples collected from 14 farms located in different agroecosystems on the northern (cooler and wetter) and southern (warmer and drier) slopes of Tenerife. Ten Trichoderma species, including T. afroharzianum, T. asperellum, T. atrobrunneum, T. gamsii, T. guizhouense, T. hamatum, T. harzianum, T. hirsutum, T. longibrachiatum, and T. virens, and two putative novel species, named T. aff. harzianum and T. aff. hortense, were identified based on the tef1-α sequences. Trichoderma virens (35.89% relative abundance) and T. aff. harzianum (27.48%) were the most abundant and dominant species on both slopes, while other species were observed only on one slope (north or south). Biodiversity indices (Margalef, Shannon, Simpson, and Pielou) showed that species diversity and evenness were highest in the healthy soils of the northern slope. The Spearman analysis showed significant correlations between Trichoderma species and soil chemistry parameters (mainly with phosphorus and soil pH). To the best of our knowledge, six species are reported for the first time in the Canary Islands (T. afroharzianum, T. asperellum, T. atrobrunneum, T. guizhouense, T. hamatum, T. hirsutum) and in the rhizosphere of banana soils (T. afroharzianum, T. atrobrunneum, T. gamsii, T. guizhouense, T. hirsutum, T. virens). This study provides essential information on the diversity/distribution of native Trichoderma species for the benefit of future applications in the control of Foc-STR4.

MaSMG7-Mediated Degradation of MaERF12 Facilitates Fusarium oxysporum f. sp. cubense Tropical Race 4 Infection in Musa acuminata.

Modern plant breeding relies heavily on the deployment of susceptibility and resistance genes to defend crops against diseases. The expression of these genes is usually regulated by transcription factors including members of the AP2/ERF family. While these factors are a vital component of the plant immune response, little is known of their specific roles in defense against Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) in banana plants. In this study, we discovered that MaERF12, a pathogen-induced ERF in bananas, acts as a resistance gene against Foc TR4. The yeast two-hybrid assays and protein-protein docking analyses verified the interaction between this gene and MaSMG7, which plays a role in nonsense-mediated RNA decay. The transient expression of MaERF12 in Nicotiana benthamiana was found to induce strong cell death, which could be inhibited by MaSMG7 during co-expression. Furthermore, the immunoblot analyses have revealed the potential degradation of MaERF12 by MaSMG7 through the 26S proteasome pathway. These findings demonstrate that MaSMG7 acts as a susceptibility factor and interferes with MaERF12 to facilitate Foc TR4 infection in banana plants. Our study provides novel insights into the biological functions of the MaERF12 as a resistance gene and MaSMG7 as a susceptibility gene in banana plants. Furthermore, the first discovery of interactions between MaERF12 and MaSMG7 could facilitate future research on disease resistance or susceptibility genes for the genetic improvement of bananas.

Codon usage bias of secretory protein in Fusarium oxysporum f. sp. cubense tropical race 4.

Banana Fusarium oxysporum f. sp. cubense tropical race 4 (Foc-TR4) is a highly destructive pathogen that infects nearly all major banana cultivars and has a tendency to spread further. Secreted proteins play a crucial role in the process of Fusarium wilt infection in bananas. In this study, we analyzed the codon usage bias (CUB) of the Foc-TR4 classical secretory protein genome for the first time and observed a strong bias toward codons ending with C. We found that 572 out of the 14,543 amino acid sequences in the Foc-TR4 genome exhibited characteristics of classical secretory proteins. The CUB was largely influenced by selection optimization pressure, as indicated by the ENC value and neutral plot analysis. Among the identified codons, such as UCC and CCC, 11 were found to be optimal for Foc-TR4 gene expression. Codons with higher GC content and a C base in the third position showed greater selectivity. The CUB in the secretory proteins encoded by Foc-TR4 provides insights into their evolutionary patterns, contributing to the development and screening of novel and effective antifungal drugs.

The Autophagy-Related Musa acuminata Protein MaATG8F Interacts with MaATG4B, Regulating Banana Disease Resistance to Fusarium oxysporum f. sp. cubense Tropical Race 4.

Banana is one of the most important fruits in the world due to its status as a major food source for more than 400 million people. Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) causes substantial losses of banana crops every year, and molecular host resistance mechanisms are currently unknown. We here performed a genomewide analysis of the autophagy-related protein 8 (ATG8) family in a wild banana species. The banana genome was found to contain 10 MaATG8 genes. Four MaATG8s formed a gene cluster in the distal part of chromosome 4. Phylogenetic analysis of ATG8 families in banana, Arabidopsis thaliana, citrus, rice, and ginger revealed five major phylogenetic clades shared by all of these plant species, demonstrating evolutionary conservation of the MaATG8 families. The transcriptomic analysis of plants infected with Foc TR4 showed that nine of the MaATG8 genes were more highly induced in resistant cultivars than in susceptible cultivars. Finally, MaATG8F was found to interact with MaATG4B in vitro (with yeast two-hybrid assays), and MaATG8F and MaATG4B all positively regulated banana resistance to Foc TR4. Our study provides novel insights into the structure, distribution, evolution, and expression of the MaATG8 family in bananas. Furthermore, the discovery of interactions between MaATG8F and MaATG4B could facilitate future research of disease resistance genes for the genetic improvement of bananas.

Dataset from transcriptome profiling of Musa resistant and susceptible cultivars in response to Fusarium oxysporum f.sp. cubense race1 and TR4 challenges using Illumina NovaSeq.

In this investigation, the study focused on the RNAseq data generated in response to Fusarium oxysporum f.sp. cubense (Foc) race1 (Cavendish infecting strain VCG 0124), targeting both resistant (cv. Rose, AA) and susceptible cultivars (Namarai, AA), and Tropical Race 4 (TR4, strain VCG 01213/16), involving resistant (cv. Rose, AA) and susceptible cultivars (Matti, AA). The respective contrasting cultivars were independently challenged with Foc race1 and TR4, and the root and corm samples were collected in two replications at varying time intervals [0th (control), 2nd, 4th, 6th, and 8th days] in duplicates. The RNA samples underwent stringent quality checks, with all 80 samples meeting the primary parameters, including a satisfactory RNA integrity number (>7). Subsequent library preparation and secondary quality control steps were executed successfully for all samples, paving the way for the sequencing phase. Sequencing generated an extensive amount of data, yielding a range of 10 to 31 million paired-end raw reads per sample, resulting in a cumulative raw data size of 11-50 GB. These raw reads were aligned against the reference genome of Musa acuminata ssp. malaccensis version 2 (DH Pahang), as well as the pathogen genomes of Foc race 1 and Foc TR4, using the HISAT2 alignment tool. The focal point of this study was the investigation of differential gene expression patterns of Musa spp. upon Foc infection. In Foc race1 resistant and susceptible root samples across the designated day intervals, a significant number of genes displayed up-regulation (ranging from 1 to 228) and down-regulation (ranging from 1 to 274). In corm samples, the up-regulated genes ranged from 1 to 149, while down-regulated genes spanned from 3 to 845. For Foc TR4 resistant and susceptible root samples, the expression profiles exhibited a notable up-regulation of genes (ranging from 31 to 964), along with a down-regulation range of 316-1315. In corm samples, up-regulated genes ranged from 57 to 929, while down-regulated genes were observed in the range of 40-936. In addition to the primary analysis, a comprehensive secondary analysis was conducted, including Gene Ontology (GO), euKaryotic Orthologous Groups (KOG) classification, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and investigations into Simple Sequence Repeats (SSRs), Single Nucleotide Polymorphisms (SNPs), and microRNA (miRNA). The complete dataset was carefully curated and housed at ICAR-NRCB, Trichy, ensuring its accuracy and accessibility for the duration of the study. Further, the raw transcriptome read datasets have been successfully submitted to the National Center for Biotechnology Information - Sequence Read Archive (NCBI-SRA) database, ensuring the accessibility and reproducibility of this valuable dataset for further research endeavors.

Secondary metabolite induced tolerance to Fusarium oxysporum f.sp. cubense TR4 in banana cv. Grand Naine through in vitro bio-immunization: a prospective research translation from induction to field tolerance.

An innovative tissue culture mediated incorporation of metabolite-based biomolecule (Bio-immune) at in vitro stage itself in banana cv. Grand Naine was developed and validated for the production of Fusarium oxysporum f.sp. cubense TR4 tolerant plantlets. The novel bio-immune formulation developed by us, exhibited a significant antifungal potency against Foc TR4 with a high percent inhibition (100%) at a 2.5% concentration of bio-immune on the 5th, 7th, and 9th DAI. Bio-immune integrated during in vitro shoot proliferation stage in banana cv. Grand Naine recorded significant enhancement in the growth of roots and shoots. Bio-immune (0.5%) fortified media produced 12.67 shoots per clump whereas control registered only 9.67 shoots per clump. Similarly, maximum root numbers (7.67) were observed in bio-immune plants which were significantly higher over control (5.0). The bio-immunized banana transplants recorded a higher survival rate (97.57%) during acclimatization as compared to the control (94.53%). Furthermore, evaluation of the bio-immunized plants in pot experiments revealed that unimmunized plants treated with FocTR4 (TF) exhibited mortality between 60 and 90 days. On the 90th day after planting, a high mean disease severity index (DSI) of 3.45 was observed with unimmunized plantlets while the bio-immunized plants (TFBI) and ICAR-FUSICONT treated plants (TFTR) showed substantially reduced DSI (0.20 and 1.00) compared to FocTR4 treated control (TF). Significant increases in polyphenol oxidase (PPO), peroxidase (POD), ß-1,3-glucanase, phenylalanine ammonia-lyase (PAL), chitinase activities, and enhanced phenol contents were recorded in bio-immunized plants compared to unimmunized plants. Field experiments at two different locations in Bihar, India revealed that bunch weight, no. of hands/bunch, and no. of fingers/hand of bio-immune treated plants were significantly higher compared to the control.

Comparative transcriptomic and weighted gene co-expression network analysis to identify the core genes in the cultivars of Musa acuminata under both infected and chemical perturbated conditions.

Banana is a high nutrient crop, which ranks fourth in terms of gross value production. Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense tropical race 4 (FocTR4), is considered the most destructive disease leading to the complete loss of production of the Cavendish cultivars Berangan, Brazilian and Williams, which are vulnerable to the infection of FocTR4. However, the treatment with benzothiadiazole, a synthetic salicylic analog, is aimed to induce resistance in plants. Thus, the treatments pertaining to the banana plants subjected to the Foc infection within the chosen cultivars were compared with chemically treated samples obtained at different time intervals for a short duration (0-4 days). The integrated omics analyses considering the parameters of WGCNA, functional annotation, and protein-protein interactions revealed that many pathways have been negatively influenced in Cavendish bananas under FocTR4 infections and the number of genes influenced also increased over time in Williams cultivar. Furthermore, elevation in immune response and resistance genes were also observed in the roots of the Cavendish banana.

Complete genome sequence of Streptomyces strain VNUA116, a potential biocontrol against phytopathogenic fusarium wilt fungus Fusarium oxysporum f. sp. cubense.

Here, we present the whole-genome sequence of Streptomyces strain VNUA116 was obtained by combining sequencing data from both PacBio RS II and DNBseq platforms. The complete circular genome is 8,306,919 bp with a GC content of 72.49%.

Pseudomonas aeruginosa Strain 91: A Multifaceted Biocontrol Agent against Banana Fusarium Wilt.

Banana Fusarium wilt (BFW), caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc), poses significant threats to banana cultivation. Currently, effective control methods are lacking, and biological control has emerged as a possible strategy to manage BFW outbreaks. In this investigation, 109 bacterial strains were isolated from the rhizospheric soil surrounding banana plants in search of potent biological agents against Foc. Strain 91 exhibited the highest antifungal activity against the causal agent of Foc and was identified as Pseudomonas aeruginosa through 16S rRNA gene sequencing and scanning electron microscopy (SEM). Elucidation of strain 91's inhibitory mechanism against Foc revealed a multifaceted antagonistic approach, encompassing the production of bioactive compounds and the secretion of cell wall hydrolytic enzymes. Furthermore, strain 91 displayed various traits associated with promoting plant growth and showed adaptability to different carbon sources. By genetically tagging with constitutively expressing GFP signals, effective colonization of strain 91 was mainly demonstrated in root followed by leaf and stem tissues. Altogether, our study reveals the potential of P. aeruginosa 91 for biocontrol based on inhibition mechanism, adaptation, and colonization features, thus providing a promising candidate for the control of BFW.

Validation and Application of a Molecular Detection System for Fusarium Wilt of Banana in China.

Fusarium wilt of banana is a devastating disease caused by Fusarium oxysporum f. sp. cubense (Foc). It has restricted the development of the banana industry worldwide and is particularly serious in China because of the large planting areas and special planting patterns. However, there is no rapid and accurate approach to detect the Foc strains that specifically occur in China because of the rich genetic diversity observed in this pathosystem. In this study, we evaluated the performance of 10 previously published PCR primer pairs on 103 representative Foc strains in China and neighboring countries and screened out a set of primers (Foc-specific primer pair SIX9-Foc-F/R, Foc R1-specific primer pair SIX6b-210-F/R, Foc R4-specific primer pair Foc-1/2, and Foc TR4-specific primer pair W2987F/R) suitable for the detection of Foc strains in China and the surrounding Southeast Asian countries. Moreover, we developed a molecular detection system to accurately identify the different physiological races of Foc. The findings of this study provide technical support for preventing and controlling the spread of Fusarium wilt of banana in the field in China.

Metatranscriptome-based strategy reveals the existence of novel mycoviruses in the plant pathogenic fungus Fusarium oxysporum f. sp. cubense.

Fusarium oxysporum f. sp. cubense (Foc) is a devastating plant pathogen that caused a great financial loss in the banana's source area. Metatranscriptomic analysis was used to determine the diversity of mycoviruses in 246 isolates of F. oxysporum f. sp. cubense. Partial or nearly complete genomes of 20 mycoviruses were obtained by BLASTp analysis of RNA sequences using the NCBI database. These 20 viruses were grouped into five distinct lineages, namely Botourmiaviridae, Endornaviridae, Mitoviridae, Mymonaviridae, Partitiviridae, and two non-classified mycoviruses lineages. To date, there is no report of the presence of mycoviruses in this pathogen. In this study, we demonstrate the presence of mycoviruses isolated from Foc. These findings enhance our overall knowledge of viral diversity and taxonomy in Foc. Further characterization of these mycoviruses is warranted, especially in terms of exploring these novel mycoviruses for innovative biocontrol of banana Fusarium wilt disease.

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.

Revealing the different resistance mechanisms of banana 'Guijiao 9' to Fusarium oxysporum f. sp. cubense tropical race 4 using comparative proteomic analysis.

Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense is a worldwide devastating fungal disease in the banana industry. The disease caused by Fusarium oxysporum f. sp. cubense is becoming more and more serious. The pathogen of Fusarium oxysporum f. sp. cubense tropical race 4 (Foc4) is the most harmful one. 'Guijiao 9' is a banana cultivar with good resistance to Foc4, which is identified by resistance screening of natural variant lines. It is of great significance to explore the resistance genes and key proteins of 'Guijiao 9' for banana cultivar improvement and disease resistance breeding. In this study, iTRAQ (isobaric Tags for Relative and Absolute quantitation) was used to analyze the xylem proteomic data of banana roots from the resistant variety 'Guijiao 9' and susceptible variety 'Williams', and the differences in protein accumulation profiles between these two varieties at 24, 48, and 72 h after infection with Foc4 were compared. The identified proteins were analyzed by the protein WGCNA (Weighted Gene Correlation Network Analysis), and the differentially expressed proteins (DEPs) were verified by qRT-PCR experiments. Proteomic analysis showed that there were differences in the protein accumulation profiles of the resistant cultivar 'Guijiao 9' and the susceptible cultivar 'Williams' after infection with Foc4, and there were differences in resistance-related proteins, biosynthesis of secondary metabolites, peroxidase, and pathogenesis-related proteins. The stress response of bananas to pathogens was affected by multiple factors. Protein co-expression analysis showed that there was a high correlation between the MEcyan module and resistance, and 'Guijiao 9' had a different resistance mechanism compared with 'Williams'. SIGNIFICANCE: 'Guijiao 9' is a banana variety with good resistance to Foc4, which is identified by screening the resistance of natural variant lines in the farmland where banana plants are seriously infected by Foc4. It is of great significance to excavate the resistance genes and key proteins of 'Guijiao 9' for banana variety improvement and disease resistance breeding. The aim of this paper is to identify the proteins and related functional modules controlling the pathogenicity differences of Foc4 by comparative proteomic analysis of 'Guijiao 9', so as to understand the resistance mechanism of banana to Fusarium wilt, and offer basis for the final identification, isolation and utilization of Foc4 resistance-related genes in banana variety improvement. The research results will also provide a basis for further understanding the host-pathogen interaction and revealing the resistance mechanism of bananas.

Loss of Phosphomannose Isomerase Impairs Growth, Perturbs Cell Wall Integrity, and Reduces Virulence of Fusarium oxysporum f. sp. cubense on Banana Plants.

Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) causes Fusarium wilt of banana, necessitating urgent measures to control this disease. However, the molecular mechanisms underlying Foc TR4 virulence remain elusive. Phosphomannose isomerase is a key enzyme involved in the biosynthesis of GDP mannose, an important precursor of fungal cell walls. In this study, two phosphomannose isomerases were identified in the Foc TR4 genome, of which only Focpmi1 was highly expressed throughout all developmental stages. Generated null mutants in Foc TR4 showed that only the ΔFocpmi1 mutant required exogenous mannose for growth, indicating that Focpmi1 is the key enzyme involved in GDP mannose biosynthesis. The Focpmi1 deficient strain was unable to grow without exogenous mannose and exhibited impaired growth under stress conditions. The mutant had reduced chitin content in its cell wall, rendering it vulnerable to cell wall stresses. Transcriptomic analysis revealed up- and down-regulation of several genes involved in host cell wall degradation and physiological processes due to the loss of Focpmi1. Furthermore, Focpmi1 was also found to be crucial for Foc TR4 infection and virulence, making it a potential antifungal target to address the threats posed by Foc TR4.

Characterization of Banana SNARE Genes and Their Expression Analysis under Temperature Stress and Mutualistic and Pathogenic Fungal Colonization.

SNAREs (soluble N-ethylmaleimide-sensitive-factor attachment protein receptors) are engines for almost all of the membrane fusion and exocytosis events in organism cells. In this study, we identified 84 SNARE genes from banana (Musa acuminata). Gene expression analysis revealed that the expression of MaSNAREs varied a lot in different banana organs. By analyzing their expression patterns under low temperature (4 °C), high temperature (45 °C), mutualistic fungus (Serendipita indica, Si) and fungal pathogen (Fusarium oxysporum f. sp. Cubense Tropical Race 4, FocTR4) treatments, many MaSNAREs were found to be stress responsive. For example, MaBET1d was up-regulate by both low and high temperature stresses; MaNPSN11a was up-regulated by low temperature but down-regulated by high temperature; and FocTR4 treatment up-regulated the expression of MaSYP121 but down-regulated MaVAMP72a and MaSNAP33a. Notably, the upregulation or downregulation effects of FocTR4 on the expression of some MaSNAREs could be alleviated by priorly colonized Si, suggesting that they play roles in the Si-enhanced banana wilt resistance. Foc resistance assays were performed in tobacco leaves transiently overexpressing MaSYP121, MaVAMP72a and MaSNAP33a. Results showed that transient overexpression of MaSYP121 and MaSNPA33a suppressed the penetration and spread of both Foc1 (Foc Race 1) and FocTR4 in tobacco leaves, suggesting that they play positive roles in resisting Foc infection. However, the transient overexpression of MaVAMP72a facilitated Foc infection. Our study can provide a basis for understanding the roles of MaSNAREs in the banana responses to temperature stress and mutualistic and pathogenic fungal colonization.

α-Pheromone Precursor Protein Foc4-PP1 Is Essential for the Full Virulence of Fusarium oxysporum f. sp. cubense Tropical Race 4.

Fusarium oxysporum f. sp. cubense (Foc), which causes Fusarium wilt of bananas, is considered one of the most destructive fungal pathogens of banana crops worldwide. During infection, Foc secretes many different proteins which promote its colonization of plant tissues. Although F. oxysporum has no sexual cycle, it has been reported to secrete an α-pheromone, which acts as a growth regulator, chemoattractant, and quorum-sensing signaling molecule; and to encode a putative protein with the hallmarks of fungal α-pheromone precursors. In this study, we identified an ortholog of the α-pheromone precursor gene, Foc4-PP1, in Foc tropical race 4 (TR4), and showed that it was necessary for the growth and virulence of Foc TR4. Foc4-PP1 deletion from the Foc TR4 genome resulted in decreased fungal growth, increased sensitivity to oxidative stress and cell-wall-damaging agents, and attenuation of pathogen virulence towards banana plantlets. Subcellular localization analysis revealed that Foc4-PP1 was concentrated in the nuclei and cytoplasm of Nicotiana benthamiana cells, where it could suppress BAX-induced programmed cell death. In conclusion, these findings suggest that Foc4-PP1 contributes to Foc TR4 virulence by promoting hyphal growth and abiotic stress resistance and inhibiting the immune defense responses of host plants.

Identification of a Major QTL-Controlling Resistance to the Subtropical Race 4 of Fusarium oxysporum f. sp. cubense in Musa acuminata ssp. malaccensis.

Vascular wilt caused by the ascomycete fungal pathogen Fusarium oxysporum f. sp. cubense (Foc) is a major constraint of banana production around the world. The virulent race, namely Tropical Race 4, can infect all Cavendish-type banana plants and is now widespread across the globe, causing devastating losses to global banana production. In this study, we characterized Foc Subtropical Race 4 (STR4) resistance in a wild banana relative which, through estimated genome size and ancestry analysis, was confirmed to be Musa acuminata ssp. malaccensis. Using a self-derived F2 population segregating for STR4 resistance, quantitative trait loci sequencing (QTL-seq) was performed on bulks consisting of resistant and susceptible individuals. Changes in SNP index between the bulks revealed a major QTL located on the distal end of the long arm of chromosome 3. Multiple resistance genes are present in this region. Identification of chromosome regions conferring resistance to Foc can facilitate marker assisted selection in breeding programs and paves the way towards identifying genes underpinning resistance.

A Network of Sporogenesis-Responsive Genes Regulates the Growth, Asexual Sporogenesis, Pathogenesis and Fusaric Acid Production of Fusarium oxysporum f. sp. cubense.

The conidia produced by Fusarium oxysporum f. sp. cubense (Foc), the causative agent of Fusarium Wilt of Banana (FWB), play central roles in the disease cycle, as the pathogen lacks a sexual reproduction process. Until now, the molecular regulation network of asexual sporogenesis has not been clearly understood in Foc. Herein, we identified and functionally characterized thirteen (13) putative sporulation-responsive genes in Foc, namely FocmedA(a), FocmedA(b), abaA-L, FocflbA, FocflbB, FocflbC, FocflbD, FocstuA, FocveA, FocvelB, wetA-L, FocfluG and Foclae1. We demonstrated that FocmedA(a), abaA-L, wetA-L, FocflbA, FocflbD, FocstuA, FocveA and Foclae1 mediate conidiophore formation, whereas FocmedA(a) and abaA-L are important for phialide formation and conidiophore formation. The expression level of abaA-L was significantly decreased in the ΔFocmedA(a) mutant, and yeast one-hybrid and ChIP-qPCR analyses further confirmed that FocMedA(a) could bind to the promoter of abaA-L during micro- and macroconidiation. Moreover, the transcript abundance of the wetA-L gene was significantly reduced in the ΔabaA-L mutant, and it not only was found to function as an activator of micro- and macroconidium formation but also served as a repressor of chlamydospore production. In addition, the deletions of FocflbB, FocflbC, FocstuA and Foclae1 resulted in increased chlamydosporulation, whereas FocflbD and FocvelB gene deletions reduced chlamydosporulation. Furthermore, FocflbC, FocflbD, Foclae1 and FocmedA(a) were found to be important regulators for pathogenicity and fusaric acid synthesis in Foc. The present study therefore advances our understanding of the regulation pathways of the asexual development and functional interdependence of sporulation-responsive genes in Foc.