Lilian Hasegawa Florentino

Hemoglobins are heme proteins and are present in some microorganisms, higher plants and mammals. In legume nodules there are two types: leghemoglobin (LegH) or symbiotic and non-symbiotic (nsHb). LegHs are present in high amounts at legumes roots and are responsible together with bacteroides for the nitrogen fixation process. Non-symbiotic hemoglobins Class 1 protein have very high affinity for O ² and are found in monocotyledons and legumes. LegH has aroused great interest in the vegetable meat industry due to its organoleptic and nutritional properties. Here, we demonstrated that soybean LegH A, C1, C2, C3 and nsHb are produced by E. coli -based cell-free protein synthesis (CFPS) and correctly synthesized in its amino acids sequence. In addition, it was also possible to reproduce some post-translational modifications confirmed by LC/MS analysis. All LegHs produced in this system showed peroxidase activity and heme binding correlated with its concentration in the assays. Furthermore, all proteins were readily digested by pepsin within 1 minute in analog digestion conditions. Therefore, LegHs and nsHb proteins were synthesized using cell-free systems (CFSs), maintaining their functionality and being digestible. These findings suggest that they could serve as viable alternative food additives for plant-based meat. Abstract Figure:

The comprehension of virus-host interactions has allowed numerous advances in developing biotechnological methodologies for plant genome editions, constituting a promising path for plant genetic engineering. Among these advancements, phage- encoded large serine-integrases have emerged as noteworthy tools to modulate plant metabolic pathways by inserting, excising, or inverting DNA stretches in a reversible and specific way. The present work shows the foundation of the Int-Plex@ (INTegrase PLant EXpression) binary memory switch system, which consists of the application of four distinct orthogonal prophage large serine-integrases (Int) (BxB1, phiC31, Int13, and Int9) as an input trigger mechanism for the inversion or excision of genomic DNA. The memory genetic switch is divided into the excision module and the inversion module. The excision module is activated by BxB1 or phiC31 enzymes (input). In this case, the DNA sequence flanked by its attachment sites is excised from the genome (output). The inversion module is activated by Int9 or Int13 (input). The inverted mgf gene sequence is flipped to its functional coding sequence, and the switch output is mGFP. Moreover, prokaryotic-based cell-free in vitro transcription-translation reactions (TxTl) were used as a fast platform for testing Ints attB/P in tandem site activity. Furthermore different plasmid delivery strategies for plant cell Int heterologous expression were tested: leaf tissue agroinfiltration of Agrobacterium tumefaciens transformed with binary plasmids and a biolistic system. After each treatment, the edited genomic DNA sequences were amplified and verified by Sanger and Nanopore sequencing. Despite the challenges of using Ints, the potential benefits are significant and deserve deeper exploration and development. The Int-Plex@ binary genome memory switch system can be applied to produce genetic circuits combined with omics tools and sgRNAs to engineer and modulate plant metabolic pathways temporally and reversibly.

Soybean is a rich source of vegetal protein for both animal and human consumption. Despite the high levels of protein in soybean seeds, industrial processing to obtain soybean bran significantly decreases the final protein content of the byproducts. To overcome this problem, cultivars with higher protein contents must be developed. However, selecting the target proteins is difficult because of the lack of information on the proteome profile of soybean bran. Therefore, this study obtained the comparative proteomic profiles of both natural coatless seeds and defatted bran from an elite tropical-soybean cultivar. Thus, their extracts were characterized using LC–MS/MS and a total of 550 proteins were identified. Among these, 526 proteins were detected in coatless seeds and 319 proteins in defatted bran. Moreover, a total of 139 proteins were identified as presenting different levels of content in coatless seeds and defatted bran. Among them, only 46 were retained after the seed processing. These proteins were clustered in several important metabolic pathways, such as amino-acid biosynthesis, sugar biosynthesis, and antioxidant activity, meaning that they could act as targets for bioactive products or genome editing to improve protein quality and quantity in soybean grains. These findings can enhance our understanding regarding protein robustness for both soybean crops and the commercial bran improvement because target proteins must remain intact after processing and must be bioactive when overexpressed. Overall, the soybean bran proteomic profile was explored for the first time, providing a valuable catalogue of target proteins that can tolerate the industrial process.

Recently, soybean consumption has increased, not only because of its potential for industrial and livestock use but also due to its beneficial effects on human health in the treatment and prevention of various diseases because soy can produce a wide number of functional proteins. Despite the soybean-producing high, elevated, nutritive and functional proteins, it also produces allergenic proteins, harmful secondary metabolites, and carcinogenic elements. So, recombinant protein systems that mimic the structures and functions of the natural proteins supply a single tunable and valuable source of advanced materials. But the availability of the technology to produce synthetic functional proteins is still limited. Therefore, Synthetic Biology is a powerful and promising science field for the development of new devices and systems able to tackle the challenges that exist in conventional studies on the development of functional protein systems. Thus, representing a new disruptive frontier that will allow better use of soybean functional proteins, both for animal and human food and for the pharmaceutical and chemistry industry.

NSP-interacting kinase (NIK1) is a receptor-like kinase identified as a virulence target of the begomovirus nuclear shuttle protein (NSP). We found that NIK1 undergoes a stepwise pattern of phosphorylation within its activation-loop domain (A-loop) with distinct roles for different threonine residues. Mutations at Thr-474 or Thr-468 impaired autophosphorylation and were defective for kinase activation. In contrast, a mutation at Thr-469 did not impact autophosphorylation and increased substrate phosphorylation, suggesting an inhibitory role for Thr-469 in kinase function. To dissect the functional significance of these results, we used NSP-expressing virus infection as a mechanism to interfere with wild type and mutant NIK1 action in plants. The NIK1 knockout mutant shows enhanced susceptibility to virus infections, a phenotype that could be complemented with ectopic expression of a 35S-NIK1 or 35S-T469A NIK1 transgenes. However, ectopic expression of an inactive kinase or the 35S-T474A NIK1 mutant did not reverse the enhanced susceptibility phenotype of knockout lines, demonstrating that Thr-474 autophosphorylation was needed to transduce a defense response to geminiviruses. Furthermore, mutations at Thr-474 and Thr-469 residues antagonistically affected NIK-mediated nuclear relocation of the downstream effector rpL10. These results establish that NIK1 functions as an authentic defense receptor as it requires activation to elicit a defense response. Our data also suggest a model whereby phosphorylation-dependent activation of a plant receptor-like kinase enables the A-loop to control differentially auto- and substrate phosphorylation.

Summary: In contrast to the accumulated data on nuclear transport mechanisms of macromolecules, little is known concerning the regulated release of nuclear‐exported complexes and their subsequent trans‐cytoplasmic movement. The bipartite begomovirus nuclear shuttle protein (NSP) facilitates the nuclear export of viral DNA and cooperates with the movement protein (MP) to transport viral DNA across the plant cell wall. Here, we identified a cellular NSP‐interacting GTPase (NIG) with biochemical properties consistent with a nucleocytoplasmic transport role. We show that NIG is a cytosolic GTP‐binding protein that accumulates around the nuclear envelope and possesses intrinsic GTPase activity. NIG interacts with NSP in vitro and in vivo (under transient expression), and redirects the viral protein from the nucleus to the cytoplasm. We propose that NIG acts as a positive contributor to geminivirus infection by modulating NSP nucleocytoplasmic shuttling and hence facilitating MP–NSP interaction in the cortical cytoplasm. In support of this, overexpression of NIG in Arabidopsis enhances susceptibility to geminivirus infection. In addition to highlighting the relevance of NIG as a cellular co‐factor for NSP function, our findings also have implications for general nucleocytoplasmic trafficking of cellular macromolecules.

The Geminiviridae family is a large family of plant viruses that has single-stranded DNA genomes and infects a large variety of crop species. In this chapter, we describe a biolistic inoculation protocol that has been successfully used to propagate new species of geminivirus in permissive hosts with total DNA extracted from infected plants. This allows us to directly investigate the biological properties of uncloned and not sap-transmissible geminiviruses.

The Begomovirus genus is the largest genus of the Geminiviridae family and comprises the whitefly transmitted geminiviruses that infect dicotyledonous plants. They can be either mono or bipartite. In this chapter, we describe the cloning of begomovirus replication modules and the subsequent functional characterization of geminivirus replication origins.