The Banana MaWRKY18, MaWRKY45, MaWRKY60 and MaWRKY70 Genes Encode Functional Transcription Factors and Display Differential Expression in Response to Defense Phytohormones.

WRKY transcription factors (TFs) play key roles in plant defense responses through phytohormone signaling pathways. However, their functions in tropical fruit crops, especially in banana, remain largely unknown. Several WRKY genes from the model plants rice (OsWRKY45) and Arabidopsis (AtWRKY18, AtWRKY60, AtWRKY70) have shown to be attractive TFs for engineering disease resistance. In this study, we isolated four banana cDNAs (MaWRKY18, MaWRKY45, MaWRKY60, and MaWRKY70) with homology to these rice and ArabidopsisWRKY genes. The MaWRKY cDNAs were isolated from the wild banana Musa acuminata ssp. malaccensis, which is resistant to several diseases of this crop and is a progenitor of most banana cultivars. The deduced amino acid sequences of the four MaWRKY cDNAs revealed the presence of the conserved WRKY domain of ~60 amino acids and a zinc-finger motif at the N-terminus. Based on the number of WRKY repeats and the structure of the zinc-finger motif, MaWRKY18 and MaWRKY60 belong to group II of WRKY TFs, while MaWRKY45 and MaWRKY70 are members of group III. Their corresponding proteins were located in the nuclei of onion epidermal cells and were shown to be functional TFs in yeast cells. Moreover, expression analyses revealed that the majority of these MaWRKY genes were upregulated by salicylic acid (SA) or methyl jasmonate (MeJA) phytohormones, although the expression levels were relatively higher with MeJA treatment. The fact that most of these banana WRKY genes were upregulated by SA or MeJA, which are involved in systemic acquired resistance (SAR) or induced systemic resistance (ISR), respectively, make them interesting candidates for bioengineering broad-spectrum resistance in this crop.

Genome-Wide Analysis of the LRR-RLP Gene Family in a Wild Banana (Musa acuminata ssp. malaccensis) Uncovers Multiple Fusarium Wilt Resistance Gene Candidates.

Banana is the most popular fruit in the world, with a relevant role in food security for more than 400 million people. However, fungal diseases cause substantial losses every year. A better understanding of the banana immune system should facilitate the development of new disease-resistant cultivars. In this study, we performed a genome-wide analysis of the leucine-rich repeat receptor-like protein (LRR-RLP) disease resistance gene family in a wild banana. We identified 78 LRR-RLP genes in the banana genome. Remarkably, seven MaLRR-RLPs formed a gene cluster in the distal part of chromosome 10, where resistance to Fusarium wilt caused by Foc race 1 has been previously mapped. Hence, we proposed these seven MaLRR-RLPs as resistance gene candidates (RGCs) for Fusarium wilt. We also identified seven other banana RGCs based on their close phylogenetic relationships with known LRR-RLP proteins. Moreover, phylogenetic analysis of the banana, rice, and Arabidopsis LRR-RLP families revealed five major phylogenetic clades shared by these plant species. Finally, transcriptomic analysis of the MaLRR-RLP gene family in plants treated with Foc race 1 or Foc TR4 showed the expression of several members of this family, and some of them were upregulated in response to these Foc races. Our study provides novel insights into the structure, distribution, evolution, and expression of the LRR-RLP gene family in bananas as well as valuable RGCs that will facilitate the identification of disease resistance genes for the genetic improvement of this crop.

The effect of benzo[a]pyrene on the gut microbiota of Nile tilapia (Oreochromis niloticus).

Benzo[a]pyrene (BaP) is a highly toxic and carcinogenic polycyclic aromatic hydrocarbon (PAH) whose toxicological effects in the gut microbiota of aquatic organisms have not yet been fully revealed. Therefore, in this study, we used high-throughput 16S rRNA gene sequencing to evaluate the effects of BaP in the gut microbiome of Oreochromis niloticus, including its possible participation in the process of detoxification and its ability to recover. The fish were injected with a single intraperitoneal dose of 20 mg kg-1 of BaP, and the effects in the microbiome were evaluated at 24, 72, and 120 h post-injection. The results indicate a clear dysbiosis (in composition, relative abundance, diversity, and interaction networks) of the gut microbiota during 24 h post-injection, dominated by Fusobacteria and Bacteroidetes and a decrease in Proteobacteria and Spirochaetae. Interestingly, a slight recovery of the microbiome begins at 72 h and stabilises at 120 h post-injection. Pathway analysis revealed the participation of the gut microbiome in PAH degradation mainly at 24 h post-injection. This study provides new insights in the toxicology of BaP in O. niloticus and the first evidence of the ability of the gut microbiome to recovery after a chemical disturbance. KEY POINTS: • Benzo[a]pyrene caused a dysbiosis in the gut microbiota of Oreochromis niloticus. • We observed an enrichment of bacteria involved in the metabolism of xenobiotics. • The gut microbiota was recovered after exposure to benzo[a]pyrene.

Transcriptomic analysis reveals key transcription factors associated to drought tolerance in a wild papaya (Carica papaya) genotype.

Most of the commercial papaya genotypes show susceptibility to water deficit stress and require high volumes of irrigation water to yield properly. To tackle this problem, we have collected wild native genotypes of Carica papaya that have proved to show better physiological performance under water deficit stress than the commercial cultivar grown in Mexico. In the present study, plants from a wild Carica papaya genotype and a commercial genotype were subjected to water deficit stress (WDS), and their response was characterized in physiological and molecular terms. The physiological parameters measured (water potential, photosynthesis, Fv/Fm and electrolyte leakage) confirmed that the papaya wild genotype showed better physiological responses than the commercial one when exposed to WDS. Subsequently, RNA-Seq was performed for 4 cDNA libraries in both genotypes (susceptible and tolerant) under well-watered conditions, and when they were subjected to WDS for 14 days. Consistently, differential expression analysis revealed that after 14 days of WDS, the wild tolerant genotype had a higher number of up-regulated genes, and a higher number of transcription factors (TF) that were differentially expressed in response to WDS, than the commercial genotype. Thus, six TF genes (CpHSF, CpMYB, CpNAC, CpNFY-A, CpERF and CpWRKY) were selected for further qRT-PCR analysis as they were highly expressed in response to WDS in the wild papaya genotype. qRT-PCR results confirmed that the wild genotype had higher expression levels (REL) in all 6 TF genes than the commercial genotype. Our transcriptomic analysis should help to unravel candidate genes that may be useful in the development of new drought-tolerant cultivars of this important tropical crop.

Enhanced production of triacylglycerols and polyunsaturated fatty acids in novel acid-tolerant mutants of the green microalga Chlorella saccharophila.

In this study, the microalga Chlorella saccharophila was subjected to ultraviolet (UV) mutagenesis, and mutant screening was conducted based on acidity tolerance to generate mutants with increased triacylglycerol (TAG) and polyunsaturated fatty acid (PUFA) contents. Two improved mutant strains (M1 and M5) were generated. M1 and M5 accumulated 27.2% and 27.4% more TAG, respectively, and showed stronger fluorescence intensity than the wild-type (WT) strain when the cells of these mutants were stained with the lipophilic Nile Red stain. In the M1 mutant, 50.5% of the fatty acid methyl esters (FAMEs) were saturated (C16:0 and C18:0) and 25.27% were monounsaturated (C18:1) fatty acids which are suitable for biofuels production. In the M5 mutant, 65.19% of the total FAMEs were nutritional PUFAs (C16:2, C18:2, and C18:3), while these FAMEs were not detected in the WT. These results demonstrated that UV mutagenesis coupled to an acid pH screening strategy represents a valuable and fast platform to generate mutants of C. saccharophila with improved TAG and PUFA contents for biofuels and nutraceutical applications, respectively.

A novel salt-inducible CrGPDH3 promoter of the microalga Chlamydomonas reinhardtii for transgene overexpression.

The expression of transgenes in the nucleus is an attractive alternative for the expression of recombinant proteins in the green microalga Chlamydomonas reinhardtii. For this purpose, a strong inducible promoter that allows protein accumulation without possible negative effects on cell multiplication and biomass accumulation is desirable. A previous study at our laboratory identified that the CrGPDH3 gene from C. reinhardtii was inducible under NaCl treatments. In this study, we cloned and characterized a 3012 bp sequence upstream of the start codon of the CrGPDH3 gene, including the 285 bp 5' untranslated region. This region was identified as the full-length promoter and named PromA (- 2727 to + 285). Deletion analysis of PromA using GUSPlus as a reporter gene enabled us to identify PromC (- 653 to + 285) as the core promoter, displaying basal expression. A region named RIA1 (- 2727 to - 1672) was suggested to contain the NaCl response elements. Moreover, deletion analysis of RIA1 enabled us to identify a region of 577 bp named RIA3 (- 2727 to - 2150) that, when cloned upstream of PromC, was able to drive the expression of GUSPlus in response to 5 and 100 mM NaCl, and 100 mM KCl, similar to the native CrGPDH3 promoter. These results expand our understanding of the transcriptional mechanism of CrGPDH3 and clearly show that CrGPDH3 promoter and its chimeric forms are highly salt-inducible and can be used as inducible promoters for the overexpression of transgenes in C. reinhardtii.

Transgenic Cavendish bananas with resistance to Fusarium wilt tropical race 4.

Banana (Musa spp.) is a staple food for more than 400 million people. Over 40% of world production and virtually all the export trade is based on Cavendish banana. However, Cavendish banana is under threat from a virulent fungus, Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) for which no acceptable resistant replacement has been identified. Here we report the identification of transgenic Cavendish with resistance to TR4. In our 3-year field trial, two lines of transgenic Cavendish, one transformed with RGA2, a gene isolated from a TR4-resistant diploid banana, and the other with a nematode-derived gene, Ced9, remain disease free. Transgene expression in the RGA2 lines is strongly correlated with resistance. Endogenous RGA2 homologs are also present in Cavendish but are expressed tenfold lower than that in our most resistant transgenic line. The expression of these homologs can potentially be elevated through gene editing, to provide non-transgenic resistance.

In silico cloning and characterization of the TGA (TGACG MOTIF-BINDING FACTOR) transcription factors subfamily in Carica papaya.

The TGA transcription factors belong to the subfamily of bZIP group D that play a major role in disease resistance and development. Most of the TGA identified in Arabidopsis interact with the master regulator of SAR, NPR1 that controls the expression of PR genes. As a first approach to determine the possible involvement of these transcription factors in papaya defense, we characterized Arabidopsis TGA orthologs from the genome of Carica papaya cv. SunUp. Six orthologs CpTGA1 to CpTGA6, were identified. The predicted CpTGA proteins were highly similar to AtTGA sequences and probably share the same DNA binding properties and transcriptional regulation features. The protein sequences alignment evidenced the presence of conserved domains, characteristic of this group of transcription factors. The phylogeny showed that CpTGA evolved into three different subclades associated with defense and floral development. This is the first report of basal expression patterns assessed by RT-PCR, from the whole subfamily of CpTGA members in different tissues from papaya cv. Maradol mature plants. Overall, CpTGA1, CpTGA3 CpTGA6 and CpTGA4 showed a basal expression in all tissues tested; CpTGA2 expressed strongly in all tissues except in petioles while CpTGA5 expressed only in petals and to a lower extent in petioles. Although more detailed studies in anthers and other floral structures are required, we suggest that CpTGA5 might be tissue-specific, and it might be involved in papaya floral development. On the other hand, we report here for the first time, the expression of the whole family of CpTGA in response to salicylic acid (SA). The expression of CpTGA3, CpTGA4 and CpTGA6 increased in response to SA, what would suggest its involvement in the SAR response in papaya.

In silico cloning and characterization of the glycerol-3-phosphate dehydrogenase (GPDH) gene family in the green microalga Chlamydomonas reinhardtii.

Glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the conversion of dihydroxyacetone phosphate (DHAP) and NADH to glycerol-3-phosphate (G3P) and NAD(+). G3P is important as a precursor for glycerol and glycerolipid synthesis in microalgae. A GPDH enzyme has been previously purified from the green microalga Chlamydomonas reinhardtii, however, no genes coding for GPDH have been characterized before. In this study, we report the in silico characterization of three putative GPDH genes from C. reinhardtii: CrGPDH1, CrGPDH2, and CrGPDH3. These sequences showed a significant similarity to characterized GPDH genes from the microalgae Dunaliella salina and Dunaliella viridis. The prediction of the three-dimensional structure of the proteins showed the characteristic fold topology of GPDH enzymes. Furthermore, the phylogenetic analysis showed that the three CrGPDHs share the same clade with characterized GPDHs from Dunaliella suggesting a common evolutionary origin and a similar catalytic function. In addition, the K(a)/K(s) ratios of these sequences suggested that they are under purifying selection. Moreover, the expression analysis showed a constitutive expression of CrGPDH1, while CrGPDH2 and CrGPDH3 were induced in response to osmotic stress, suggesting a possible role for these two sequences in the synthesis of glycerol as a compatible solute in osmoregulation, and perhaps also in lipid synthesis in C. reinhardtii. This study has provided a foundation for further biochemical and genetic studies of the GPDH family in this model microalga, and also opportunities to assess the potential of these genes to enhance the synthesis of TAGs for biodiesel production.

The green microalga Chlorella saccharophila as a suitable source of oil for biodiesel production.

The aim of this study was to investigate the potential of the green microalga Chlorella saccharophila as a source of oil for biodiesel production. We evaluated for the first time, the effect of salinity and/or nitrogen depletion (ND) on cell growth, lipid accumulation and lipid profile in this microalga. The fatty acid methyl esters (FAME) identified for C. saccharophila in this study consisted of C-16:0, C-18:0, C-18:1 cis, and C-18:1 trans. Among these, C-18:1 (indicator of biodiesel quality) was the main FAME found, representing approximately 76 and 80% of total FAME under normal and ND growing conditions, respectively. Under a normal growing condition this microalga showed 154.63 mg l(-1) d(-1), 63.33 mg l(-1) d(-1), and 103.73 mg l(-1) of biomass productivity, lipid productivity, and FAME yield, respectively. The higher biomass productivity (159.58 mg l(-1) d(-1)), lipid productivity (99.33 mg l(-1) d(-1)), and FAME yield (315.53 mg l(-1)) were obtained under the ND treatment. In comparison to other related studies, our results suggest that C. saccharophila can be considered as a suitable source of oil for biodiesel production.

The in vitro secretome of Mycosphaerella fijiensis induces cell death in banana leaves.

The hemibiotrophic filamentous fungus Mycosphaerella fijiensis causes the banana foliar disease known as black Sigatoka, responsible for major worldwide losses in the banana fruit industry. In this work the in vitro secretome of M. fijiensis was characterized. Native and denaturant polyacrylamide gel protease assays showed the M. fijiensis secretome contains protease activity capable of degrading gelatin. Necrotic lesions on leaves were produced by application of the in vitro secretome to the surface of one black Sigatoka-resistant banana wild species, one susceptible cultivar and the non-host plant Carica papaya. To distinguish if necrosis by the secretome is produced by phytotoxins or proteins, the latter ones were precipitated with ammonium sulfate and applied in native or denatured forms onto leaves of the same three plant species. Proteins applied in both preparations were able to produce necrotic lesions. Application of Pronase, a commercial bacterial protease suggested that the necrosis was, at least in part, caused by protease activity from the M. fijiensis secretome. The ability to cause necrotic lesions between M. fijiensis secreted- and ammonium sulfate-precipitated proteins, and purified lipophilic or hydrophilic phytotoxins, was compared. The results suggested that leaf necrosis arises from the combined action of non-host specific hydrolytic activities from the secreted proteins and the action of phytotoxins. This is the first characterization of the M. fijiensis protein secretome produced in vitro but, more importantly, it is also the first time the M. fijiensis secretome has been shown to contain virulence factors capable of causing necrosis to its natural host.

Structural and phylogenetic analysis of Pto-type disease resistance gene candidates in banana.

The tomato Pto gene encodes a serine/threonine kinase (STK) whose molecular characterization has provided valuable insights into the disease resistance mechanism of tomato and it is considered as a promising candidate for engineering broad-spectrum pathogen resistance in this crop. In this study, a pair of degenerate primers based on conserved subdomains of plant STKs similar to the tomato Pto protein was used to amplify similar sequences in banana. A fragment of approximately 550 bp was amplified, cloned and sequenced. The sequence analysis of several clones revealed 13 distinct sequences highly similar to STKs. Based on their significant similarity with the tomato Pto protein (BLASTX E value <3e-53), seven of them were classified as Pto resistance gene candidates (Pto-RGCs). Multiple sequence alignment of the banana Pto-RGC products revealed that these sequences contain several conserved subdomains present in most STKs and also several conserved residues that are crucial for Pto function. Moreover, the phylogenetic analysis showed that the banana Pto-RGCs were clustered with Pto suggesting a common evolutionary origin with this R gene. The Pto-RGCs isolated in this study represent a valuable sequence resource that could assist in the development of disease resistance in banana.