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Maize lethal necrosis is a destructive virus disease of maize caused by maize chlorotic mottle virus (MCMV) in combination with a virus in the family Potyviridae. Emergence of MLN is typically associated with the introduction of MCMV or its vectors and understanding its spread through seed is critical for disease management. Previous studies suggest that although MCMV is detected on seed, the seed transmission rate of this virus is low. However, mechanisms influencing its transmission are poorly understood. Elucidating these mechanisms is crucial for informing strategies to prevent spread on contaminated seed. In this study, we evaluated the rate of MCMV seed transmission using seed collected from plants that were artificially inoculated with MCMV isolates from Hawaii and Kenya. Grow-out tests indicated that MCMV transmission through seed was rare, with a rate of 0.004% among the more than 85,000 seed evaluated, despite detection of MCMV at high levels in the seed lots. To understand factors that limit transmission from seed, MCMV distribution in seed tissues was examined using serology and immunolocalization. The virus was present at high levels in maternal tissues, the pericarp and pedicel, but absent from filial endosperm and embryo seed tissues. The ability to transmit MCMV from seed to uninfected plants was tested to evaluate virus viability. Transmission was negatively associated with both seed maturity and moisture content. Transmission of MCMV from infested seed dried to less than 15% moisture was not detected, suggesting proper handling could be important for minimizing spread of MCMV through seed. [ABSTRACT FROM AUTHOR]

Maize lethal necrosis (MLN) is a devastating synergistic viral disease of maize caused by maize chlorotic mottle virus (MCMV) plus a virus in the Potyviridae family. The disease has severely impacted maize production in East Africa, Asia, and South America over the past decade, and the presence of MCMV and maize infecting potyviruses in the United States is an ongoing concern. In this study, we performed a genome‐wide association study (GWAS) using the Maize 282 Association Mapping Panel (AMP) to characterize the genetic architecture of host response to MCMV and to identify disease‐tolerant lines. The GWAS analysis detected genomic variants on chromosomes 3, 4, 5, and 8 that were significantly associated with response to MCMV. Several lines in the 282 AMP were highly tolerant to infection, including CML333, one of the parental lines used in the maize nested association mapping population. Quantitative trait locus analysis of the B73 × CML333 recombinant inbred line population revealed an MCMV tolerance locus on chromosome 10. These findings further our understanding of the genetic architecture of MCMV tolerance and will facilitate the development of maize lines, hybrids, and cultivars with improved resistance to MLN and MCMV. Core Ideas: A GWAS identified seven maize chlorotic mottle virus (MCMV) tolerance loci in the Maize 282 Association Mapping Panel.Maize lines with strong MCMV tolerance, including CML333, were identified.A MCMV tolerance QTL was mapped in the B73 × CML333 RIL population.New MCMV tolerant lines and tolerance loci will facilitate development of improved maize. [ABSTRACT FROM AUTHOR]

Background: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has become a powerful tool for functional genomics in plants. The RNA-guided nuclease can be used to not only generate precise genomic mutations, but also to manipulate gene expression when present as a deactivated protein (dCas9). Results: In this study, we describe a vector toolkit for analyzing dCas9-mediated activation (CRISPRa) or inactivation (CRISPRi) of gene expression in maize protoplasts. An improved maize protoplast isolation and transfection method is presented, as well as a description of dCas9 vectors to enhance or repress maize gene expression. Conclusions: We anticipate that this maize protoplast toolkit will streamline the analysis of gRNA candidates and facilitate genetic studies of important trait genes in this transformation-recalcitrant plant. [ABSTRACT FROM AUTHOR]

Maize lethal necrosis (MLN) is emergent in East Africa, first reported in 2011 in Kenya, and is devastating to maize production in the region. MLN is caused by coinfection of maize with the emergent maize chlorotic mottle virus (MCMV) and any of several maize‐infecting potyviruses endemic in East Africa and worldwide. Here, we examined the distribution of MCMV and sugarcane mosaic virus (SCMV), the major viruses contributing to MLN in Rwanda. These and other viruses in maize across Rwanda were further characterized by deep sequencing. When identified, MCMV had high titres and minimal sequence variability, whereas SCMV showed moderate titres and high sequence variability. Deep sequencing also identified maize streak virus and other maize‐associated viruses, including a previously described polerovirus, maize yellow mosaic virus, and barley yellow dwarf virus, diverse maize‐associated totiviruses, maize‐associated pteridovirus, Zea mays chrysovirus 1, and a maize‐associated betaflexivirus. Detection of each virus was confirmed in maize samples by reverse transcription polymerase chain reaction. [ABSTRACT FROM AUTHOR]

The effects of cytoplasmic protein synthesis and protein phosphatase activity on NADH:nitrate reductase (NR) activity, protein and transcript were examined in maize (Zea mays L.) seedling leaves. A rapid increase in NR activity, measured in the presence of 5 mM Mg2+, was found upon exposure of excised leaves to light. Inhibitors of protein phosphatase activity (okadaic acid [OKA] and microcystin [MC]-LR) completely prevented the increase in NR activity. The cytoplasmic protein synthesis inhibitor, cycloheximide (CHX). did not affect Mg2+ inhibition of NR activity during the dark-to-light transition. Vmax NR activity, measured in the presence of Pi and EDTA, remained constant or increased slightly in maize leaves during the first 2 h of the light period. OKA, MC-LR or CHX treatment caused a 40 to 50% reduction in Vmax NR activity during this time. Incorporation of 35S-Met into NR protein was reduced more than 90% by CHX and 80% by OKA. The inhibition of NR protein synthesis by CHX and OKA correlated with a 50 to 60% decrease in 35S-Met incorporation into total soluble protein over the treatment period. The increase in NR mRNA levels early in the light period was prevented by OKA and MC-LR, but not by CHX. OKA had a similar effect on sucrose phosphate synthase mRNA levels, but did not affect Catalase 1 or Catalase 3 mRNA accumulation. The data suggest that light-induced decreases in Mg2+ inhibition of NR activity and transcript levels are independent of new protein synthesis. The effects of OKA and MC-LR indicate that protein phosphatase activities could be involved, directly or indirectly, in the regulation of NR activity, protein synthesis and transcript accumulation. [ABSTRACT FROM AUTHOR]