Camila Perdoncini Carvalho

Soybean (Glycine max) is relatively recalcitrant to genetic manipulations; hence, it is often interrogated with transient means such as virus-induced gene silencing (VIGS). We earlier modified cowpea severe mosaic virus (CPSMV) to develop a soybean-friendly VIGS system referred to as QUIN-FZ. Here we report additional calibrations of this system. We enhanced the intra-bacterial stability of plasmid QUIN, which contained a CPSMV RNA1 cDNA embedded with four introns, by adding a fifth intron, resulting in PENTIN. We separately upgraded the plasmid FZ, which contained a modified CPSMV RNA2 cDNA with a cloning site in the middle of the viral polyprotein, by creating another cloning site within the 3′ untranslated region, leading to ZY. We next used the new PENTIN-ZY system to investigate a putative soybean protein kinase designated QL18. Virus-mediated overexpression of two allelic, 147-amino-acid (aa) protein fragments, derived from two different QL18 orthologs, elicited drastically different responses in soybeans. While the fragment derived from soybean accession OX20-8 prevented the cognate virus from infecting top young leaves in at least 50% of inoculated seedlings, its allelic counterpart derived from soybean accession PI427105B elicited apical necrosis in 100% of soybean seedlings. By examining progeny viruses as well as viruses encoding chimeras of the two fragments, we identified more than a dozen mutations that abrogated these unique phenotypes. Our findings establish the PENTIN-ZY system as a versatile tool for overexpressing small proteins and protein fragments, accelerating their functional characterization.

Soybean (Glycine max) is relatively recalcitrant to genetic manipulations, hence is often interrogated with transient means such as virus-induced gene silencing (VIGS). We earlier modified cowpea severe mosaic virus (CPSMV) to develop a soybean-friendly VIGS system referred to as QUIN-FZ. Here we report additional calibrations of this system. We enhanced the intra-bacterial stability of plasmid QUIN, which contained a CPSMV RNA1 cDNA embedded with four introns, by adding a fifth intron, resulting in PENTIN. We separately upgraded the plasmid FZ, which contained a modified CPSMV RNA2 cDNA with a cloning site in the middle of the viral polyprotein, by creating another cloning site within the 3’ untranslated region, leading to ZY. We next used the new PENTIN-ZY system to investigate a putative soybean protein kinase designated QL18. Virus-mediated overexpression of two allelic, 147-amino-acid (aa) protein fragments, derived from two different QL18 orthologs, elicited drastically different responses in soybeans. While the fragment derived from soybean accession OX20-8 prevented the cognate virus from infecting top young leaves in at least 50% of inoculated seedlings, its allelic counterpart derived from soybean accession PI427105B elicited apical necrosis in 100% of soybean seedlings. By examining progeny viruses as well as viruses encoding chimeras of the two fragments, we identified more than a dozen mutations that abrogated these unique phenotypes. Our findings establish the PENTIN-ZY system as a versatile tool for overexpressing small proteins and protein fragments, accelerating their functional characterization.

Reproductively lethal mutations (RLMs) are mutations that, upon expression of the encoded lethal phenotypes, cause individuals carrying them to die or to be sterile. An underappreciated fact is that loss-of-function RLMs in protein-coding genes can be phenotypically shielded by favorable environments rendering these genes conditionally non-essential, or by their sister alleles in diploid organisms. Absent of rigorous mitigation, such phenotype-shielding causes the number of genes incurring RLMs to increase over time, and simultaneously allows each RLM to reach high allele frequencies in a conspecific population. Over-accumulation of RLMs then sets the population up for eventual concurrent expression of large numbers of RLMs, and massive deaths in rapid succession, possibly even population-level extinction. This hypothetical scenario in turn predicts that organismal lineages that evolved means to minimize the allele frequencies of phenotypically shielded RLMs are favored by natural selection. We argue that bottlenecking the genome copies destined for reproduction is a universal strategy adopted by all living beings to compel phenotype-based RLM purging. We further postulate that primitive RNA replicons must first evolve bottlenecked reproduction before evolving the capacity to encode diffusible products. In more complex, multicellular organisms, RLM management through bottlenecked reproduction gains additional reinforcement through sexual reproduction. In short, the evidence chronicled in this essay strongly suggest that the Bottleneck, Isolate, Amplify, Select (BIAS) principle, originally proposed to explain intracellular evolutionary dynamics of viruses, may be universally applicable to all living beings.

The impact of Gibberella ear rot (GER; caused by Fusarium graminearum) on deoxynivalenol contamination of grain (DON) and yield components in maize were investigated using data from 30 environments in Ohio (3 years x 10 locations). Fifteen hybrids, later classified as susceptible (SU), moderately susceptible (MS), or moderately resistant (MR), based on the magnitude of differences in mean arcsine square-root-transformed GER severity (arcSEV) and log-transformed DON (logDON) relative to a reference susceptible check, were planted in each environment, and 10 ears per hybrid were inoculated with a spore suspension of F. graminearum. Relationships between GER severity and DON were well described by a Kono-Sugino-type nonlinear equation. Estimated parameters representing height (A) and steepness (β) of the curves were significantly higher for SU than MS and MR hybrids, but A was not significantly different between MS and MR. Results from a surrogacy analysis showed that GER was a moderate trial- and individual-level surrogate for DON. Both grain weight per ear and ear diameter decreased with increasing arcSEV, but the regression slopes varied among resistance classes. The rates of reduction in both yield components per unit increase in arcSEV were significantly greater for SU than for MS and MR. An estimated 50% reduction in grain weight occurred at 62% GER severity for SU, compared to 77% severity for MS, and 83% for MR. These results show that GER severity can be used as a surrogate for early estimation of DON contamination and yield loss to help guide grain handling and marketing decisions.

Bacterial resistance is a sanitary issue explained by indiscriminate use of nonprescription drugs, and antimicrobial use in food production for growth promotion. Bothropstoxin-I (BthTx-I) is a phospholipase A2 (PLA2) from Bothrops jararacussu venom, which has a known antimicrobial effect. The goal of this study was the unprecedented evaluation of in vivo antimicrobial activity of BthTx-I in broilers. Microbiological, biochemical, and histological parameters were determined using 84 21-day old broilers that were kept in cages with four birds each at a density of 625 cm2/broiler. The experiment was randomized by three treatments with seven repetitions of four broilers each that lasted seven days. The treatments were: 1) bacitracin zinc diet; 2) PLA2-BthTx-I; 3) without additives. The data obtained from the studied variables was subjected to analysis of variance and an F-test at the 5% significance level. Averages of each variable in each treatment were compared by Tukey’s test. Broiler bacterial cloacal counts showed that BthTx-I decreased the microbial population without reducing body weight, intestinal morphology, or liver or kidney histopathological damage. The toxin showed in vivo activity, being an alternative for better performance in the production of broiler chickens, because it acted by decreasing the microbial load of potentially pathogenic bacteria in the intestinal microbiota of the birds and did not cause muscle, liver or kidney damage at the assessed dosage.