Identification of mimp-associated effector genes in Fusarium oxysporum f. sp. cubense race 1 and race 4 and virulence confirmation of a candidate effector gene.

Effectors secreted by microbes contribute to pathogen virulence and/or avirulence on host plants in the interaction of plants and microbes. Also, the effector repertoire determines the host specificity of a pathogen. Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of banana wilt; however, knowledge about Foc effector genes is very limited. In this study, genome-wide effector gene identification was performed in Foc race 1 (Foc 1) and Foc race 4 (Foc 4) based on the context association between the effector genes and the transposable element mimp. A total of 20 candidate effector genes were identified, of which 3 were Foc 1-specific, 6 were Foc 4-specific, and 11 were present in both Foc 1 and Foc 4. Most genes (14 out of 20) showed a significant transcriptional burst in planta compared with in-culture conditions, from more than 10-fold to 1,617-fold, and at the highest 32,725-fold. In addition to Foc 1- and Foc 4-specific genes, the genes Foc 283, Foc 495, and Foc 594 also exhibited transcriptional race specificity. Sixteen of the twenty genes were predicted to have a signal peptide, nine genes might encode real effectors predicted by EffectorP 2.0, and eight genes had predicted motifs. To validate the pathogenicity of the candidate effector genes, we generated knockout mutant and complementants of the gene Foc 1324 and tested their virulence on banana plants. The results showed that Foc 1324 was a virulent factor and required for the pathogenicity of Foc 4.

Identification and characterization of miRNA169 family members in banana (Musa acuminata L.) that respond to fusarium oxysporum f. sp. cubense infection in banana cultivars.

MicroRNAs (miRNAs) play an important role in plant resistance to pathogen infections. However, little is known about the role of miRNAs in banana Fusarium wilt, which is the most economically devastating disease in banana production. In the present study, we identified and characterized a total of 18 miR169 family members in banana (Musa acuminata L.) based on small RNA sequencing. The banana miR169 family clustered into two groups based on miRNA evolutionary analysis. Multiple sequence alignment indicated a high degree of sequence conservation in miRNA169 family members across 28 plant species. Computational target prediction algorithms were used to identify 25 targets of miR169 family members in banana. These targets were enriched in various metabolic pathways that include the following molecules: glycine, serine, threonine, pentose, glycerolipids, nucleotide sugars, starch, and sucrose. Through miRNA transcriptomic analysis, we found that ma-miR169a and ma-miR169b displayed high expression levels, whereas the other 16 ma-miR169 members exhibited low expression in the HG and Baxi banana cultivars. Further experiments indicate that there were negative relationships between ma-miR169a, ma-miR169b and their targets basing on their expression levels to Foc4 (Fusarium oxysporum f. sp. cubense tropical race 4) infection in resistant cultivars. But they were low expressed in susceptive cultivars. These results suggested that the expression levels of ma-miR169a and ma-miR169b were consistent with the resistance degree of the banana cultivars to Foc4. The analysis presented here constitutes a starting point to understand ma-miR169-mediated Fusarium wilt resistance at the transcriptional level in banana and predicts possible candidate targets for the genetic improvement of banana resistance to Foc4.

Geographical and Genetic Divergence Among Papaya ringspot virus Populations Within Hainan Province, China.

Papaya ringspot virus (PRSV) severely affects the global papaya industry. Transgenic papaya has been proven to have effective resistance to PRSV isolates from Hawaii, Thailand, Taiwan, and other countries. However, those transgenic cultivars failed to show resistance to Hainan Island isolates. Some 76 PRSV samples, representative of all traditional papaya planting areas across five cities (Wen Chang, n = 13; Cheng Mai, n = 14; Chang Jiang, n = 11; Le Dong, n = 25; and San Ya, n = 13) within Hainan Province, were investigated. Results revealed three genetic diversity groups (Hainan I, II, and III) that correlated with geographical distribution. Frequent mutations among PRSV isolates from Hainan were also observed. The high genetic divergence in PRSV isolates from Hainan is likely to be the cause of the failure of genetically modified papaya that targets sequence-specific virus.

Proteomics of Fusarium oxysporum race 1 and race 4 reveals enzymes involved in carbohydrate metabolism and ion transport that might play important roles in banana Fusarium wilt.

Banana Fusarium wilt is a soil-spread fungal disease caused by Fusarium oxysporum. In China, the main virulence fungi in banana are F. oxysporum race 1 (F1, weak virulence) and race 4 (F4, strong virulence). To date, no proteomic analyses have compared the two races, but the difference in virulence between F1 and F4 might result from their differentially expressed proteins. Here we report the first comparative proteomics of F1 and F4 cultured under various conditions, and finally identify 99 protein species, which represent 59 unique proteins. These proteins are mainly involved in carbohydrate metabolism, post-translational modification, energy production, and inorganic ion transport. Bioinformatics analysis indicated that among the 46 proteins identified from F4 were several enzymes that might be important for virulence. Reverse transcription PCR analysis of the genes for 15 of the 56 proteins revealed that their transcriptional patterns were similar to their protein expression patterns. Taken together, these data suggest that proteins involved in carbohydrate metabolism and ion transport may be important in the pathogenesis of banana Fusarium wilt. Some enzymes such as catalase-peroxidase, galactosidase and chitinase might contribute to the strong virulence of F4. Overexpression or knockout of the genes for the F4-specific proteins will help us to further understand the molecular mechanism of Fusarium-induced banana wilt.

Genome and transcriptome analysis of the fungal pathogen Fusarium oxysporum f. sp. cubense causing banana vascular wilt disease.

BACKGROUND: The asexual fungus Fusarium oxysporum f. sp. cubense (Foc) causing vascular wilt disease is one of the most devastating pathogens of banana (Musa spp.). To understand the molecular underpinning of pathogenicity in Foc, the genomes and transcriptomes of two Foc isolates were sequenced. METHODOLOGY/PRINCIPAL FINDINGS: Genome analysis revealed that the genome structures of race 1 and race 4 isolates were highly syntenic with those of F. oxysporum f. sp. lycopersici strain Fol4287. A large number of putative virulence associated genes were identified in both Foc genomes, including genes putatively involved in root attachment, cell degradation, detoxification of toxin, transport, secondary metabolites biosynthesis and signal transductions. Importantly, relative to the Foc race 1 isolate (Foc1), the Foc race 4 isolate (Foc4) has evolved with some expanded gene families of transporters and transcription factors for transport of toxins and nutrients that may facilitate its ability to adapt to host environments and contribute to pathogenicity to banana. Transcriptome analysis disclosed a significant difference in transcriptional responses between Foc1 and Foc4 at 48 h post inoculation to the banana 'Brazil' in comparison with the vegetative growth stage. Of particular note, more virulence-associated genes were up regulated in Foc4 than in Foc1. Several signaling pathways like the mitogen-activated protein kinase Fmk1 mediated invasion growth pathway, the FGA1-mediated G protein signaling pathway and a pathogenicity associated two-component system were activated in Foc4 rather than in Foc1. Together, these differences in gene content and transcription response between Foc1 and Foc4 might account for variation in their virulence during infection of the banana variety 'Brazil'. CONCLUSIONS/SIGNIFICANCE: Foc genome sequences will facilitate us to identify pathogenicity mechanism involved in the banana vascular wilt disease development. These will thus advance us develop effective methods for managing the banana vascular wilt disease, including improvement of disease resistance in banana.

Extraction of high-quality RNA from rubber tree leaves.

A specific technique capable of producing high-quality RNA for rapid amplification of cDNA ends (RACE) was established for challenging tissues: leaves of the rubber tree. Total RNA was extracted by cetyltrimethylammonium bromide (CTAB)-LiCl combined with TRIzol reagent. The isolated RNA was highly intact. With RNA as template, full-length cDNA was obtained (NCBI, AY461413) by RACE.

Three new compounds from soil actinomycete Streptomyces albospinus 15-4-2.

Three new compounds, 2-methyl-2,5,6-bornantriol (1), 4,4'-(3-hydroxypropane-1,1-diyl)diphenol (2), and 7-(4-methoxybenzyl)-4,5,6,7-tetrahydro-1,3-oxazepine-5,6-diol (3), were isolated from the fermentation broth of the soil actinomycete Streptomyces albospinus 15-4-2. Their structures were completely elucidated using the combination of 1D, 2D NMR techniques (COSY, HMQC, HMBC, and ROESY), and HR-ESI-MS analysis. None of the compounds 1-3 showed any inhibitory effect on Fusarium oxysporum f.sp. cubense race 4.

Extraction of Teucrium manghuaense and evaluation of the bioactivity of its extract.

The ethanol extract of Teucrium manghuaense grown in Hainan province (China) was analysed by GC and GC/MS. Of the constituents 84-96% were identified on the basis of their GC retention times and their mass spectra in regard to authentic compounds. The results revealed that it contained 9-methyl-9-azabicyclo[4.2.1]nonane (7.43%), 2-methylpyrrolidine (19.42%), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (10.84%), and squalene (28.55%), as major components, constituting 66.24% of the extract. The chemical characterization of the ethanol extract by GC/MS also allowed identification of 1-octen-3-one (3.41%), 2-pentyl-piperidine (2.25%), 1-(2-methyl-1-propenyl)-piperidine (4.63%), 2,2'-diethoxy-5,5'-bi-1-pyrroline (0.41%), (Z,Z,Z)-9,12,15-octadecatrieniic acid, 2,3-dihydroxypropyl ester (1.56%), vitamin E (2.95%) and stigmasterol (1.39%). Finally, the antioxidant and anti-tumor activities of the ethanol extract have been evaluated. Results show that the extract of Teucrium manghuaense leaf possesses strong DPPH(-), hydroxyl radical scavenging activity and anti-tumor activity.