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Recent reverse genetics technologies have enabled genetic manipulation of plant negative‐strand RNA virus (NSR) genomes. Here, we report construction of an infectious clone for the maize‐infecting Alphanucleorhabdovirus maydis, the first efficient NSR vector for maize. The full‐length infectious clone was established using agrobacterium‐mediated delivery of full‐length maize mosaic virus (MMV) antigenomic RNA and the viral core proteins (nucleoprotein N, phosphoprotein P, and RNA‐directed RNA polymerase L) required for viral transcription and replication into Nicotiana benthamiana. Insertion of intron 2 ST‐LS1 into the viral L gene increased stability of the infectious clone in Escherichia coli and Agrobacterium tumefaciens. To monitor virus infection in vivo, a green fluorescent protein (GFP) gene was inserted in between the N and P gene junctions to generate recombinant MMV‐GFP. Complementary DNA (cDNA) clones of MMV‐wild type (WT) and MMV‐GFP replicated in single cells of agroinfiltrated N. benthamiana. Uniform systemic infection and high GFP expression were observed in maize inoculated with extracts of the infiltrated N. benthamiana leaves. Insect vectors supported virus infection when inoculated via feeding on infected maize or microinjection. Both MMV‐WT and MMV‐GFP were efficiently transmitted to maize by planthopper vectors. The GFP reporter gene was stable in the virus genome and expression remained high over three cycles of transmission in plants and insects. The MMV infectious clone will be a versatile tool for expression of proteins of interest in maize and cross‐kingdom studies of virus replication in plant and insect hosts. [ABSTRACT FROM AUTHOR]

Background Insects are the most important epidemiological factors for plant virus disease spread, with >75% of viruses being dependent on insects for transmission to new hosts. The black-faced leafhopper (Graminella nigrifrons Forbes) transmits two viruses that use different strategies for transmission: Maize chlorotic dwarf virus (MCDV) which is semi-persistently transmitted and Maize fine streak virus (MFSV) which is persistently and propagatively transmitted. To date, little is known regarding the molecular and cellular mechanisms in insects that regulate the process and efficiency of transmission, or how these mechanisms differ based on virus transmission strategy. Results RNA-Seq was used to examine transcript changes in leafhoppers after feeding on MCDV-infected, MFSV-infected and healthy maize for 4 h and 7 d. After sequencing cDNA libraries constructed from whole individuals using Illumina next generation sequencing, the Rnnotator pipeline in Galaxy was used to reassemble the G. nigrifrons transcriptome. Using differential expression analyses, we identified significant changes in transcript abundance in G. nigrifrons. In particular, transcripts implicated in the innate immune response and energy production were more highly expressed in insects fed on virus-infected maize. Leafhoppers fed on MFSV-infected maize also showed an induction of transcripts involved in hemocoel and cell-membrane linked immune responses within four hours of feeding. Patterns of transcript expression were validated for a subset of transcripts by quantitative real-time reverse transcription polymerase chain reaction using RNA samples collected from insects fed on healthy or virus-infected maize for between a 4 h and seven week period. Conclusions We expected, and found, changes in transcript expression in G. nigrifrons feeding of maize infected with a virus (MFSV) that also infects the leafhopper, including induction of immune responses in the hemocoel and at the cell membrane. The significant induction of the innate immune system in G. nigrifrons fed on a foregut-borne virus (MCDV) that does not infect leafhoppers was less expected. The changes in transcript accumulation that occur independent of the mode of pathogen transmission could be key for identifying insect factors that disrupt vector-mediated plant virus transmission. [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]

Nitrate is the most common N source used by higher plants and is the ‘substrate inducer’ of nitrate and nitrite reductase activities. However, environmental nitrate also has other effects on higher plants, which include the induction of a high affinity nitrate transport system, changes in root morphology, and increased root respiration. Nitrate induces nitrate and nitrite reductase activities by altering gene expression, mainly by enhancing transcription of the respective genes. We have begun to view these responses of higher plant tissues to environmental nitrate in the context of a model for signal transduction. In our model, nitrate is a signal for developmental changes in the physiology of the plant, which are expressed in a hierarchy. The primary responses include induction of genes for nitrate and nitrite reductases, nitrate uptake and translocation systems, and DNA regulatory proteins required for expression of the secondary response gene systems. The secondary responses include more complex phenomena such as proliferation of the root system, enhancement of respiration and other changes in the physiology of the plant. [ABSTRACT FROM AUTHOR]

Abstract: Mechanisms for genetic resistance to diseases caused by potyviruses, especially Maize dwarf mosaic virus (MDMV), in maize (Zea mays L.) remain unclear. Previous studies indicated that MDMV can replicate in and spread to groups of cells in maize leaves of the virus resistant inbred line Pa405, but systemic movement of the virus did not occur. In this study, the responses of Pa405 and the virus-susceptible inbred line Oh28 were compared in germinating maize embryos. Four days post inoculation (dpi), MDMV was detected immunocytochemically in 45% of resistant and 19% of susceptible shoots, respectively. Systemic movement of MDMV out of inoculated leaves occurred between 2 and 4 dpi in Oh28 seedlings. Microarray analysis was used to examine the effects of MDMV inoculation on gene expression in shoots of resistant and susceptible lines at 4 dpi. Approximately 15% of transcripts were differentially expressed between the resistant and susceptible lines. Of the transcripts with >10 fold greater expression in one line, more than 70% were up-regulated in the resistant line. Substantially fewer genes were differentially expressed in MDMV- and mock-inoculated shoots, with 21 and 30 genes being differentially regulated in the susceptible and resistant lines, respectively. For a subset of genes, differential expression was validated using quantitative real-time PCR. There was no clustering of differentially expressed transcripts in the previously identified QTLs important for MDMV resistance. These results provide a short list of virus responsive transcripts to begin to explain the resistance response in Pa405 versus the susceptible response in Oh28. [Copyright &y& Elsevier]