Optimizing RNAi-Target by Nicotiana benthamiana-Soybean Mosaic Virus System Drives Broad Resistance to Soybean Mosaic Virus in Soybean.

Soybean mosaic virus (SMV) is a prevalent pathogen of soybean (Glycine max). Pyramiding multiple SMV-resistance genes into one individual is tedious and difficult, and even if successful, the obtained multiple resistance might be broken by pathogen mutation, while targeting viral genome via host-induced gene silencing (HIGS) has potential to explore broad-spectrum resistance (BSR) to SMV. We identified five conserved target fragments (CTFs) from S1 to S5 using multiple sequence alignment of 30 SMV genome sequences and assembled the corresponding target-inverted-repeat constructs (TIRs) from S1-TIR to S5-TIR. Since the inefficiency of soybean genetic transformation hinders the function verification of batch TIRs in SMV-resistance, the Nicotiana benthamiana-chimeric-SMV and N. benthamiana-pSMV-GUS pathosystems combined with Agrobacterium-mediated transient expression assays were invented and used to test the efficacy of these TIRs. From that, S1-TIR assembled from 462 bp CTF-S1 with 92% conservation rate performed its best on inhibiting SMV multiplication. Accordingly, S1-TIR was transformed into SMV-susceptible soybean NN1138-2, the resistant-healthy transgenic T1-plants were then picked out via detached-leaf inoculation assay with the stock-plants continued for progeny reproduction (T1 dual-utilization). All the four T3 transgenic progenies showed immunity to all the inoculated 11 SMV strains under individual or mixed inoculation, achieving a strong BSR. Thus, optimizing target for HIGS via transient N. benthamiana-chimeric-SMV and N. benthamiana-pSMV-GUS assays is crucial to drive robust resistance to SMV in soybean and the transgenic S1-TIR-lines will be a potential breeding source for SMV control in field.

The NF-Y-PYR module integrates the abscisic acid signal pathway to regulate plant stress tolerance.

Drought and salt stresses impose major constraints on soybean production worldwide. However, improving agronomically valuable soybean traits under drought conditions can be challenging due to trait complexity and multiple factors that influence yield. Here, we identified a nuclear factor Y C subunit (NF-YC) family transcription factor member, GmNF-YC14, which formed a heterotrimer with GmNF-YA16 and GmNF-YB2 to activate the GmPYR1-mediated abscisic acid (ABA) signalling pathway to regulate stress tolerance in soybean. Notably, we found that CRISPR/Cas9-generated GmNF-YC14 knockout mutants were more sensitive to drought than wild-type soybean plants. Furthermore, field trials showed that overexpression of GmNF-YC14 or GmPYR1 could increase yield per plant, grain plumpness, and stem base circumference, thus indicating improved adaptation of soybean plants to drought conditions. Taken together, our findings expand the known functional scope of the NF-Y transcription factor functions and raise important questions about the integration of ABA signalling pathways in plants. Moreover, GmNF-YC14 and GmPYR1 have potential for application in the improvement of drought tolerance in soybean plants.

Development of dual-emission cluster of Ag atoms for genetically modified organisms detection.

A DNA-silver nanocluster with two distinct emissions is devised, in which this unique modality has been exploited to develop a novel nanosensor for transgenic DNA detection. TEM and fluorescence analysis revealed the formation of Ag nanoclusters with a size of around 2 nm, which exhibit dual-emissions at 550 nm (green) and 630 nm (red). Moreover, in the presence of the target sequence (CaMV 35S promoter) from the transgenic plant, the nanoclusters showed an enhancement in the green emission and a reduction in the red emission. This property provided a ratiometric-sensing platform which lacks unavoidable noises. The ratio of green to red fluorescence emission (G/R) of the nanoclusters exhibited a linear relation with the target concentration in the range 10 to 1000 nM. However, the control DNA did not affect this ratio, which clearly confirmed the selective response of the designed nanosensor. This sensing platform had a detection limit of 1.5 nM and identified the DNA of transgenic soybeans within a short time. The mechanistic evaluation of the nanoclusters further revealed the role of protonated cytosine bases in the dual emission behavior. Finally, unique features of the designed nanosensor may improve the current approaches for the development and manufacturing of GMO detection tools.

Genomic characterization of plant cell wall degrading enzymes and in silico analysis of xylanases and polygalacturonases of Fusarium virguliforme.

BACKGROUND: Plant cell wall degrading enzymes (PCWDEs) are a subset of carbohydrate-active enzymes (CAZy) produced by plant pathogens to degrade plant cell walls. To counteract PCWDEs, plants release PCWDEs inhibitor proteins (PIPs) to reduce their impact. Several transgenic plants expressing exogenous PIPs that interact with fungal glycoside hydrolase (GH)11-type xylanases or GH28-type polygalacturonase (PG) have been shown to enhance disease resistance. However, many plant pathogenic Fusarium species were reported to escape PIPs inhibition. Fusarium virguliforme is a soilborne pathogen that causes soybean sudden death syndrome (SDS). Although the genome of F. virguliforme was sequenced, there were limited studies focused on the PCWDEs of F. virguliforme. Our goal was to understand the genomic CAZy structure of F. viguliforme, and determine if exogenous PIPs could be theoretically used in soybean to enhance resistance against F. virguliforme. RESULTS: F. virguliforme produces diverse CAZy to degrade cellulose and pectin, similar to other necrotorphic and hemibiotrophic plant pathogenic fungi. However, some common CAZy of plant pathogenic fungi that catalyze hemicellulose, such as GH29, GH30, GH44, GH54, GH62, and GH67, were deficient in F. virguliforme. While the absence of these CAZy families might be complemented by other hemicellulases, F. virguliforme contained unique families including GH131, polysaccharide lyase (PL) 9, PL20, and PL22 that were not reported in other plant pathogenic fungi or oomycetes. Sequence analysis revealed two GH11 xylanases of F. virguliforme, FvXyn11A and FvXyn11B, have conserved residues that allow xylanase inhibitor protein I (XIP-I) binding. Structural modeling suggested that FvXyn11A and FvXyn11B could be blocked by XIP-I that serves as good candidate for developing transgenic soybeans. In contrast, one GH28 PG, FvPG2, contains an amino acid substitution that is potentially incompatible with the bean polygalacturonase-inhibitor protein II (PvPGIP2). CONCLUSIONS: Identification and annotation of CAZy provided advanced understanding of genomic composition of PCWDEs in F. virguliforme. Sequence and structural analyses of FvXyn11A and FvXyn11B suggested both xylanases were conserved in residues that allow XIP-I inhibition, and expression of both xylanases were detected during soybean roots infection. We postulate that a transgenic soybean expressing wheat XIP-I may be useful for developing root rot resistance to F. virguliforme.

Desempenho fotossintético e micorrização em plantas de soja Roundup Ready tratadas com diferentes formulações de glyphosate / Photosynthetic performance and mycorrhization in Roundup Ready soybean plants treated with different formulations of glyphosate

Com a expansão do cultivo de soja resistente ao glyphosate, observa-se aumento considerável tanto do uso desse herbicida como do número de formulações comerciais à base deste princípio ativo. Objetivou-se, portanto, avaliar o efeito de seis formulações de glyphosate (Roundup Original®, Trop®, Roundup Ultra®, Roundup WG®, Roundup Transorb R® e Zapp Qi®) em parâmetros fotossintéticos, colonização micorrízica e produtividade de soja (TMG 125 Roundup Ready), na região do Alto Paranaíba. Utilizou-se do delineamento em blocos casualizados, com quatro repetições. Uma única aplicação dos herbicidas (720 g e.a. ha-1) foi realizada entre os estádios V2-V3. Aos 3 e 7 dias após a aplicação dos tratamentos (DAT), foram feitas avaliações instantâneas de trocas gasosas entre 08:00 e 9:00 h, em folíolos completamente expandidos. Em seguida, o desempenho fotossintético foi avaliado por meio de curvas de luz (taxa de assimilação líquida de CO2 vs irradiância). A colonização micorrízica, altura de plantas e massa seca da parte aérea foram avaliadas quando as plantas de soja atingiram o estádio R2. Além disso, avaliaram-se o peso de sementes e produtividade de soja. As diferentes formulações de glyphosate não afetaram as trocas gasosas das plantas de soja, nem a eficiência dessas plantas na utilização da irradiância, razão pela qual também não se observou diferenças significativas na massa seca da parte aérea, colonização micorrízica e produtividade de soja.

The expansion of soybean resistant to glyphosate has caused considerable increase in the use of this herbicide as well as the number of formulations based on this active ingredient. The aim is evaluate the effect of six formulations of glyphosate (Roundup Original®, Trop®, Roundup Ultra®, Roundup WG®, Roundup Transorb R® e Zapp Qi®) in photosynthetic parameters, mycorrhizal colonization and soybean yield (TMG 125 Roundup Ready), in the Alto Paranaíba. It was used a randomized block design with four replications. A single application of herbicides (780 g a.e ha-1) was performed between stages V2-V3. At 3 and 7 days after application of the treatments were evaluated instantaneous gas exchange between 08:00 and 9:00 am, in fully expanded leaflets. Then, the photosynthetic performance was evaluated by means of light curves (CO2 assimilation rate vs. irradiance). The mycorrhizal colonization, plant height and shoot dry matter were assessed when the soybean plants reached the stage R2. In addition, we evaluated the weight of seeds and soybean yield. The different formulations of glyphosate did not affect gas exchange of soybean plants and the efficiency of these plants in the use of irradiance, therefore also no observed significant differences in shoot dry weight, mycorrhizal colonization and soybean yield.