Site-specific single amino acid changes to Lys or Arg in the central region of the movement protein of a hybrid bromovirus are required for adaptation to a nonhost.

A hybrid Cowpea chlorotic mottle virus (CCMV) [CCMV(B3a)] in which the CCMV 3a movement protein gene is replaced by the 3a (B3a) gene of Brome mosaic virus cannot infect cowpea systemically. Previously, analysis of RNA3 cDNA clones constructed from cowpea-adapted mutants derived from CCMV(B3a) revealed that a single codon change in the B3a gene allowed CCMV(B3a) to infect cowpea systemically. In this study, to extend the analysis of the CCMV(B3a) adaptation mechanism, we directly sequenced B3a gene RT-PCR products prepared from 28 cowpea plants in which cowpea-adapted mutants appeared, and found seven patterns of a codon change localized at five specific positions in the central region (Ser(118), Glu(132), Glu(138), Gln(178), and Ser(180)). All of the patterns involved an amino acid change to Lys or Arg. Mutational analysis of the B3a gene demonstrated that a single codon change resulting in either Lys or Arg at any of the five positions was sufficient for the adaptation of CCMV(B3a) to cowpea. In contrast, CCMV(B3a) variants with a codon change resulting in Lys or Arg at three other positions (137, 155, and 161) in the B3a gene not only showed lack of systemic infection of cowpea but also showed weakened initial cell-to-cell movement in the inoculated leaves and diminished B3a accumulation in protoplasts. These results suggest that adaptive changes in the B3a gene are site-specifically selected in cowpea plants.

Novel fungal transcriptional activators, Cmr1p of Colletotrichum lagenarium and pig1p of Magnaporthe grisea, contain Cys2His2 zinc finger and Zn(II)2Cys6 binuclear cluster DNA-binding motifs and regulate transcription of melanin biosynthesis genes in a developmentally specific manner.

Colletotrichum lagenarium and Magnaporthe grisea are plant pathogenic fungi that produce melanin during the appressorial differentiation stage of conidial germination and during the late stationary phase of mycelial growth. Here, we report the identification of genes for two unique transcription factors, CMR1 (Colletotrichum melanin regulation) and PIG1 (pigment of Magnaporthe), that are involved in melanin biosynthesis. Both Cmr1p and Pig1p contain two distinct DNA-binding motifs, a Cys2His2 zinc finger motif and a Zn(II)2Cys6 binuclear cluster motif. The presence of both these motifs in a single transcriptional regulatory protein is unique among known eukaryotic transcription factors. Deletion of CMR1 in C. lagenarium caused a defect in mycelial melanization, but not in appressorial melanization. Also, cmr1Delta mutants do not express the melanin biosynthetic structural genes SCD1 and THR1 during mycelial melanization, although the expression of these two genes was not affected during appressorial melanization.

Bromovirus movement protein conditions for the host specificity of virus movement through the vascular system and affects pathogenicity in cowpea.

Previously, we reported that CCMV(B3a), a hybrid of bromovirus Cowpea chlorotic mottle virus (CCMV) with the 3a cell-to-cell movement protein (MP) gene replaced by that of cowpea-nonadapted bromovirus Brome mosaic virus (BMV), can form small infection foci in inoculated cowpea leaves, but that expansion of the foci stops between 1 and 2 days postinoculation. To determine whether the lack of systemic movement of CCMV(B3a) is due to restriction of local spread at specific leaf tissue interfaces, we conducted more detailed analyses of infection in inoculated leaves. Tissue-printing and leaf press-blotting analyses revealed that CCMV(B3a) was confined to the inoculated cowpea leaves and exhibited constrained movement into leaf veins. Immunocytochemical analyses to examine the infected cell types in inoculated leaves indicated that CCMV(B3a) was able to reach the bundle sheath cells through the mesophyll cells and successfully infected the phloem cells of 50% of the examined veins. Thus, these data demonstrate that the lack of long-distance movement of CCMV(B3a) is not due to an inability to reach the vasculature, but results from failure of the virus to move through the vascular system of cowpea plants. Further, a previously identified 3a coding change (A776C), which is required for CCMV(B3a) systemic infection of cowpea plants, suppressed formation of reddish spots, mediated faster spread of infection, and enabled the virus to move into the veins of inoculated cowpea leaves. From these data, and the fact that CCMV(B3a) directs systemic infection in Nicotiana benthamiana, a permissive systemic host for both BMV and CCMV, we conclude that the bromovirus 3a MP engages in multiple activities that contribute substantially to host-specific long-distance movement through the phloem.

Chloroplast-encoded protein as a subunit of acetyl-CoA carboxylase in pea plant.

The gene product of an open reading frame of the chloroplast genome, accD, that has sequence similarity with a subunit of acetyl-CoA carboxylase from Escherichia coli was detected immunochemically in pea chloroplasts. The apparent molecular mass of the accD protein was 87 kDa on SDS-polyacrylamide gel electrophoresis. The protein was acidic and had less mobility than the calculated value, 67,116. Acetyl-CoA carboxylase activity solubilized from pea chloroplasts was inhibited by antibodies against recombinant accD protein. The antibodies precipitated a polypeptide of 35 kDa containing biotin and a polypeptide of 91 kDa together with the 87-kDa-accD protein. The accD protein formed a complex with the molecular mass of about 700 kDa, probably with the 35- and 91-kDa proteins. These results indicate that the chloroplast-encoded polypeptide, accD protein, is a component of a functional acetyl-CoA carboxylase in chloroplasts and this enzyme is a multi-subunit complex, like that from E. coli. The synthesis of accD protein was not induced by light.