Accumulation of Maize chlorotic dwarf virus proteins in its plant host and leafhopper vector

AbstractThe genome of Maize chlorotic dwarf virus (MCDV; genus Waikavirus; family Sequiviridae) consists of a monopartite positive-sense RNA genome encoding a single large polyprotein. Antibodies were produced to His-fusions of three undefined regions of the MCDV polyprotein: the N-terminus of the polyprotein (R78), a region between coat proteins (CPs) and the nucleotide-binding site (NBS) (R37), and a region between the NBS and a 3C-like protease (R69). The R78 antibodies react with proteins of 50 kDa (P50), 35 kDa (P35), and 25 kDa (P25) in virus preparations, and with P35 in plant extracts. In extracts of the leafhopper vector Graminella nigrifrons fed on MCDV-infected plants, the R78 antibodies reacted with P25 but not with P50 and P35. The R69 antibodies bound proteins of approximately 36 kDa (P36), 30 kDa (P30), and 26 kDa (P26) in virus preparations, and P36 and P26 in plant extracts. Antibodies to R37 reacted with a 26-kDa protein in purified virus preparations, but not in plant extracts. Neither the R69 nor the R37 antibodies bound any proteins in G. nigrifrons. Thus, in addition to the three CPs, cysteine protease and RNA-dependent RNA polymerase, the MCDV polyprotein is apparently post-transitionally cleaved into P50, P35, P25, P36, P30, and P26

Evaluación de la resistencia genética de híbridos de maíz al virus del mosaico de la caña de azúcar (SCMV)

La resistencia genética es la manera más eficiente de controlar las enfermedades ya que no tiene un costo adicional para el agricultor y no contamina el ambiente. Un cultivo de maíz sano es la regla, la excepción es la enfermedad. El primer paso en todo programa de mejoramiento genético para incorporar resistencia al cultivo es identificar materiales resistentes entre las variedades tradicionales y comerciales, poblaciones mejoradas de alta diversidad genética (pooles), colecciones núcleo y colecciones para estudios genéticos. Para esto es necesario realizar evaluaciones en condiciones controladas donde se inocula el patógeno y se le brindan las condiciones para que la planta se enferme. En caso de enfermedades virales, las técnicas de inoculación incluyen el frotamiento, punción vascular y la utilización de los vectores naturales que transmiten el virus. Sugarcane mosaic virus (SCMV) está entre los principales virus que afectan al cultivo de maíz en los valles altos y trópicos de Latinoamérica y alrededor del mundo. En la Estación Experimental Litoral Sur del INIAP se inocularon 32 híbridos comerciales y experimentales de maíz utilizando la técnica del frotamiento en plántulas, con un aislamiento de SCMV colectado en la Estación Experimental Portoviejo del INIAP. Dos semanas después de la inoculación se evaluó la incidencia de los síntomas de la enfermedad en las hojas nuevas. El experimento tuvo tres réplicas biológicas en un diseño  completo al azar, con 20 plantas por tratamiento. No existieron híbridos resistentes, lo que indica la necesidad de incorporar genes de resistencia a SCMV en las poblaciones de mejoramiento genético de maíces tropicales de grano amarillo duro de las empresas públicas y privadas

The Capsid Protein of \u3ci\u3eTurnip Crinkle Virus\u3c/i\u3e Overcomes Two Separate Defense Barriers to Facilitate Systemic Movement of the Virus in \u3ci\u3eArabidopsis\u3c/i\u3e

The capsid protein (CP) of Turnip crinkle virus (TCV) is a multifunctional protein needed for virus assembly, suppression of RNA silencing-based antiviral defense, and long-distance movement in infected plants. In this report, we have examined genetic requirements for the different functions of TCV CP and evaluated the interdependence of these functions. A series of TCV mutants containing alterations in the CP coding region were generated. These alterations range from single-amino-acid substitutions and domain truncations to knockouts of CP translation. The latter category also contained two constructs in which the CP coding region was replaced by either the cDNA of a silencing suppressor of a different virus or that of green fluorescent protein. These mutants were used to infect Arabidopsis plants with diminished antiviral silencing capability (dcl2 dcl3 dcl4 plants). There was a strong correlation between the ability of mutants to reach systemic leaves and the silencing suppressor activity of mutant CP. Virus particles were not essential for entry of the viral genome into vascular bundles in the inoculated leaves in the absence of antiviral silencing. However, virus particles were necessary for egress of the viral genome from the vasculature of systemic leaves. Our experiments demonstrate that TCV CP not only allows the viral genome to access the systemic movement channel through silencing suppression but also ensures its smooth egress by way of assembled virus particles. These results illustrate that efficient long-distance movement of TCV requires both functions afforded by the CP

The Capsid Protein of \u3ci\u3eTurnip Crinkle Virus\u3c/i\u3e Overcomes Two Separate Defense Barriers to Facilitate Systemic Movement of the Virus in \u3ci\u3eArabidopsis\u3c/i\u3e

The capsid protein (CP) of Turnip crinkle virus (TCV) is a multifunctional protein needed for virus assembly, suppression of RNA silencing-based antiviral defense, and long-distance movement in infected plants. In this report, we have examined genetic requirements for the different functions of TCV CP and evaluated the interdependence of these functions. A series of TCV mutants containing alterations in the CP coding region were generated. These alterations range from single-amino-acid substitutions and domain truncations to knockouts of CP translation. The latter category also contained two constructs in which the CP coding region was replaced by either the cDNA of a silencing suppressor of a different virus or that of green fluorescent protein. These mutants were used to infect Arabidopsis plants with diminished antiviral silencing capability (dcl2 dcl3 dcl4 plants). There was a strong correlation between the ability of mutants to reach systemic leaves and the silencing suppressor activity of mutant CP. Virus particles were not essential for entry of the viral genome into vascular bundles in the inoculated leaves in the absence of antiviral silencing. However, virus particles were necessary for egress of the viral genome from the vasculature of systemic leaves. Our experiments demonstrate that TCV CP not only allows the viral genome to access the systemic movement channel through silencing suppression but also ensures its smooth egress by way of assembled virus particles. These results illustrate that efficient long-distance movement of TCV requires both functions afforded by the CP

Virus-Induced Gene Silencing in Diverse Maize Lines Using the Brome Mosaic Virus-based silencing vector

Virus-induced gene silencing (VIGS) is a widely used tool for gene function studies in many plant species, though its use in cereals has been limited. In addition, within cereal species the varieties that best respond during VIGS screens are often not known. Using a Brome mosaic virus (BMV) vector designed to silence the maize phytoene desaturase (PDS) gene, a genetically diverse set of maize inbred lines was screened for development of gene silencing after inoculation of seeds through the novel use of a vascular puncture inoculation technique. In addition to Va35, which previously was shown to support silencing, maize lines NC300, Ki11, Oh7b, M162W and CML52 displayed significant visible photobleaching when challenged with the BMV-PDS. In these plants, targeted PDS mRNA expression was decreased 50-80% relative to levels in plants that were inoculated with BMV containing a fragment of the GUS gene or were mock-inoculated

Nitrate regulation of the oxidative pentose phosphate pathway in maize (Zea mays L.) root plastids: Induction of 6-phosphogluconate dehydrogenase activity, protein and transcript levels

We examined the effect of nitrate on the expression of the NADPH producing enzymes of the oxidative pentose phosphate pathway, glucose-6- phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in maize seedlings (Zea mays L. W64A x A182E). In extracts of 5 day old maize roots and leaves treated with 10 mM KNO3, G6PDH and 6PGDH activities increased by 44 and 53%, respectively, relative to untreated roots. In isolated plastids from KNO3 treated roots, G6PDH and 6PGDH specific activities were more than 25- and 12-fold higher than in the untreated control. Western blot analysis showed higher levels of 6PGDH protein in root plastid extracts from KNO3 treated plants. The data suggest that KNO3 specifically affects the plastidic forms of G6PDH and 6PGDH. Three classes of 6PGDH cDNA were identified in maize roots. Of these, one cDNA hybridized with a transcript that accumulated rapidly and transiently in response to low concentrations of external nitrate. The accumulation of this transcript was not affected by pretreating plants with 50 μg/ml cycloheximide, which was previously shown to inhibit cytoplasmic protein synthesis in maize roots by more than 85% (Gowri et al., Plant Mol. Biol. 26 (1998) 679). Neither NH4+ nor K+ treatment affected transcript accumulation. The data indicate coordinated regulation of genes and enzymes required for NO3- assimilation and NADPH production in root plastids