Functional analysis of soybean PR-10 genes

Soybean (Glycine max) is the most important oilseed source and one of the most important crops worldwide. In 2014, 249 million metric tons were produced across the world. Even though the worldwide production is increasing every year, scientists, farmers and companies are still struggling to match production increases, to increase in world population growth. The challenge is not simply to produce more, but also to produce more on the same area of land. A main limit to increasing crop yields is the variety of diseases, mainly bacterial and fungal. It is estimated that around 15% of the crop production every year is lost due to biological threats. Understanding how soybean responds in defense to pathogens at a molecular level would help produce innovated seeds and plants that can better withstand biological attacks. This research was designed to characterize a soybean gene family that responds to multiple pathogens within a few hours of infection, the PR-10 gene family. We identified six members of the PR-10 gene family based on expression patterns from in-house microarray studies. Gene-specific PCR primers were designed to clone full-length cDNA of selected PR-10 genes. The cDNA were sequence verified and transferred into an Agrobacterium overexpression vector, expression controlled by the CaMV35S constitutive promoter. Four PR-10 family members were transformed into Arabidopsis thaliana, and alterations in defense responses were monitored in the PR-10 transformants. Two RNAi constructs were made for future transformation into soybean, to silence these genes and ascertain their function in soybean defense. Additionally, the response of a PR-10 promoter was assayed by studying GFP expression controlled by a PR-10 promoter versus the constitutive promoter GmUbi. These research results increase our understanding of PR-10 function and verify the effectiveness of PR-10 in defense response to pathogen infection, which could potentially lead to the development of markers that are associated with pathogen resistance, and also provide genetic material for basic research and possible development of transgenics with enhanced resistance

Evaluating the Efficiency in the Application of Transformation and CRISPR/Cas9 Gene-editing Technique on Pumpkins

With the simplicity of a unique genome engineering mechanism, CRISPR/Cas9 gene-editing technique has amazed scholars with its effectiveness and efficiency in manipulating gene sequences.[1] As this advanced technique develops, its applications on different species arise as prominent subjects yet to be determined. Due to the great economic value of pumpkins and the need for examining CRISPR/Cas9 gene-editing efficiency, Casperita pumpkin (Cucurbita pepo) is chosen as the subject to be investigated on. Through introducing CRISPR/Cas9 system —for modifying phytoene desaturase (PDS) gene— into pumpkin seeds with Agrobacterium-mediated transformation, we regenerate transgenic pumpkins and expect to observe albino leaves in the transformed plants. By identifying mutated pumpkins and analyzing genotyping data, the efficiency in the application of transformation and CRISPR/Cas9 gene-editing technique on pumpkins can be established. Utilizing the findings, we aim to make contribution in developing an effective, promising gene-editing practice for pumpkin and maximizing its benefits in agriculture

Bacterial spot of tomato and pepper: insights into host-pathogen interactions

The Solanaceae family, containing approximately 100 genera and 2,500 species, is the third most valuable crop family in the world, and the most valuable vegetable crop. Some of the most economically important Solanaceae are tomato (Solanum lycopersicum), pepper (Capsicum spp.), eggplant (S. melongena) and tobacco (Nicotiana tabacum). Bacterial infection and subsequent disease symptoms cause enormous yield losses to crops in this family. Xanthomonas spp., the causal agent of bacterial spot disease, can result in millions of dollars in losses of solanaceous crop production worldwide. It is almost impossible to control the disease once it is present in the plant. Therefore, it is necessary to have a better understanding of host-pathogen interactions at the molecular level to detail how some of the Xanthomonas and host molecules interact. Increasingly, new evidence has demonstrated variability in the epitope regions of bacterial flagellin, including in regions harboring the microbe-associated molecular patterns flg22 and flgII-28 that are recognized by the pattern recognition receptors FLS2 and FLS3, respectively. Additionally, since bacterial motility is known to contribute to pathogen virulence in early stages of infection and chemotaxis, reductions in or loss of motility can significantly reduce bacterial fitness. In Chapter 2, I observed variation in the motility for many isolates, irrespective of their flagellin sequence. Instead, I determined that past growth conditions may have a significant impact on the motility status of isolates, as we could minimize this variability by inducing motility using chemoattractant assays. Additionally, motility could be significantly suppressed under nutrient-limited conditions, and bacteria could “remember” its prior motility status after storage at ultra-cold temperatures. While some flagellin variants may impart altered motility, external growth parameters and gene expression regulation appear to have more significant impacts on the motility phenotypes for these Xanthomonas species. In Chapter 3, I determined that variations in flg22 and flgII-28 epitopes led to differential recognition by the immune systems in tomato, chili pepper, and bell pepper plants. One variant of flgII-28 had a polymorphism that allowed it to evade recognition by tomato and chili pepper plants; however, the FLS3 variant present in bell pepper plants recognizes this flgII-28 variant, as shown by differential immune responses in the production of reactive oxygen species and in the inhibition of seedling growth. In tomato and chili pepper plants, I observed larger bacterial populations of strains with flagellin variants predicted not to be recognized by either FLS2 and FLS3, suggesting that these bacteria can evade flagellin recognition. However, there were no difference in bacterial populations in bell pepper plants, indicating that chili pepper plants possess an FLS3 receptor that can recognize all flagellin variants tested. Overall, this flgII-28 polymorphism allows some, but not all, Xanthomonas species to evade both FLS2-and FLS3-mediated oxidative burst responses, and impacts the virulence of the bacteria after infection into different plant hosts. In Chapter 4, I wanted to further investigate the FLS3 polymorphisms that may be causing differential immune recognition of flgII-28 in tomato, chili, and bell pepper plants. I first obtained the FLS3 sequence information from chili and bell pepper plants that showed polymorphic amino acids at 12 residues located in different parts of the FLS3 proteins. I then performed in silico predictions to identify a region that may potentially function as the binding pocket of the receptor and further narrowed down my findings to two putative amino acid polymorphisms. Finally, I found charge and surface accessibility predictions in the flgII-28 epitope that is not recognized by tomato and chili pepper FLS3 proteins that may influence its binding to FLS3. In conclusion, I anticipate that these results will improve our understanding of host-pathogen interactions between Xanthomonas species and tomato and pepper plants, specifically at the initial events in pattern-triggered immunity. I hope that these findings could be used in the development of genetically engineered crops with enhanced resistance to bacterial diseases.U of I OnlyAuthor requested U of Illinois access only (OA after 2yrs) in Vireo ETD syste

Aplicação de aminoácidos e micronutrientes na produção de feijão (Phaseolus vulgaris L.).

Aiming to verify the effects of the use of sources of amino acids associated to micronutrients on the final yield of common bean, was conducted an experiment at FAZU in Uberaba-MG, using Carioca kind of bean, cultivar Pérola. The design was in randomized blocks with eight treatments and four repetitions. The sowing was done in August 18, 2008, the final stand of 240.000 plants ha-1.  Fertilization was held with 8-28-16 and coverage with urea. The treatments were constituted of T1: witness; T2: seed treatment (B: 0,1%; Cu: 0,1%; Mo: 2,%; Zn: 4,6%); T3: seed treatment + foliar fertilization at 25 DAE (B: 0,3%; Mn: 2,%; Mo: 1%; Zn: 3% + amino acids); T4: seed treatment + foliar application of amino acids at 25 DAE; T5: seed treatment + foliar application of amino aciads in the pre and post-bloom (40 and 50 DAE); T6: foliar fertilization at 25 DAE (B: 0,3%; Mn: 2%; Mo: 1,%; Zn: 3% + amino acids); T7: foliar application of amino acids at 25 DAE; T8: foliar application (B: 0,3%; Mn: 2%; Mo: 1,%; Zn: 3% + amino acids) in the pre and post-bloom (40 and 50 DAE). It was evaluated: final yield, number of pods/plants, number of grains/pods and the mass of 100 grains. The results did not show relevant difference.  Com o objetivo de verificar os efeitos do uso de fontes de aminoácidos associados aos micronutrientes no rendimento final do feijoeiro, foi conduzido um experimento na FAZU em Uberaba-MG, utilizando o feijão Carioca, cultivar Pérola. O delineamento foi em blocos casualizados, com oito tratamentos e quatro repetições. A semeadura foi realizada em 18 de agosto de 2008, no plantio final de 240.000 plantas ha-1. A fertilização foi realizada com 8-28-16 e cobertura com uréia. Os tratamentos foram constituídos por T1: testemunha; T2: tratamento de sementes (B: 0,1%; Cu: 0,1%; Mo: 2,%; Zn: 4,6%); T3: tratamento de sementes + adubação foliar aos 25 DAE (B: 0,3%; Mn: 2,%; Mo: 1%; Zn: 3% + aminoácidos); T4: tratamento de sementes + aplicação foliar de aminoácidos aos 25 DAE; T5: tratamento de sementes + aplicação foliar de aminoácidos no pré e pós-florescimento (40 e 50 DAE); T6: adubação foliar aos 25 DAE (B: 0,3%; Mn: 2%; Mo: 1,%; Zn: 3% + aminoácidos); T7: aplicação foliar de aminoácidos aos 25 DAE; T8: aplicação foliar (B: 0,3%; Mn: 2%; Mo: 1,%; Zn: 3% + aminoácidos) no pré e pós-florescimento (40 e 50 DAE). Foram avaliados: produção final, número de vagens / plantas, número de grãos / vagens e massa de 100 grãos. Os resultados não mostraram diferença relevante