H. F. Li

HC-Pro protein of sugar cane mosaic virus interacts specifically with maize ferredoxin-5 in vitro and in planta

Symptom development of a plant viral disease is a result of molecular interactions between the virus and its host plant; thus, the elucidation of specific interactions is a prerequisite to reveal the mechanism of viral pathogenesis. Here, we show that the chloroplast precursor of ferredoxin-5 (Fd V) from maize (Zea mays) interacts with the multifunctional HC-Pro protein of sugar cane mosaic virus (SCMV) in yeast, Nicotiana benthamiana cells and maize protoplasts. Our results demonstrate that the transit peptide rather than the mature protein of Fd V precursor could interact with both N-terminal (residues 1-100) and C-terminal (residues 301-460) fragments, but not the middle part (residues 101-300), of HC-Pro. In addition, SCMV HC-Pro interacted only with Fd V, and not with the other two photosynthetic ferredoxin isoproteins (Fd I and Fd II) from maize plants. SCMV infection significantly downregulated the level of Fd V mRNA in maize plants; however, no obvious changes were observed in levels of Fd I and Fd II mRNA. These results suggest that SCMV HC-Pro interacts specifically with maize Fd V and that this interaction may disturb the post-translational import of Fd V into maize bundle-sheath cell chloroplasts, which could lead to the perturbation of chloroplast structure and function.

Influence of cytoplasmic heat shock protein 70 on viral infection of Nicotiana benthamiana

The accumulation of heat shock protein 70 (Hsp70) generally occurs in plants infected with viruses. However, the effect of Hsp70 accumulation on plant viral infection and pathogenesis remains elusive. In this study, the expression of six Hsp70 genes was found to be induced by the four diverse RNA viruses, Tobacco mosaic virus, Potato virus X (PVX), Cucumber mosaic virus and Watermelon mosaic virus, in Nicotiana benthamiana. Heat treatment enhanced the accumulation and systemic infection of these viruses. Similar results were obtained for viral infection in plants heterologously expressing an Arabidopsis cytoplasmic Hsp70 through either a PVX vector or Agrobacterium infiltration. In contrast, viral infection was compromised in cytoplasmic NbHsp70c-1 gene-silenced plants. These data demonstrate that the cytoplasmic Hsp70s can enhance the infection of N. benthamiana by diverse viruses.

Plastocyanin Transit Peptide Interacts with Potato virus X Coat Protein, While Silencing of Plastocyanin Reduces Coat Protein Accumulation in Chloroplasts and Symptom Severity in Host Plants

Potato virus X coat protein (PVXCP) is, through communication with host proteins, involved in processes such as virus movement and symptom development. Here, we report that PVXCP also interacts with the precursor of plastocyanin, a protein involved in photosynthesis, both in vitro and in vivo. Yeast two-hybrid analysis indicated that PVXCP interacted with only the plastocyanin transit peptide. In subsequent bimolecular fluorescence complementation assays, both proteins were collocated within chloroplasts. Western blot analyses of chloroplast fractions showed that PVXCP could be detected in the envelope, stroma, and lumen fractions. Transmission electron microscopy demonstrated that grana were dilated in PVX-infected Nicotiana benthamiana. Furthermore, virus-induced gene silencing of plastocyanin by prior infection of N. benthamiana using a Tobacco rattle virus vector reduced the severity of symptoms that developed following subsequent PVX infection as well as the accumulation of PVXCP in isolated chloroplasts. However, PVXCP could not be detected in pea chloroplasts in an in vitro re-uptake assay using the plastocyanin precursor protein. Taken together, these data suggest that PVXCP interacts with the plastocyanin precursor protein and that silencing the expression of this protein leads to reduced PVXCP accumulation in chloroplasts and ameliorates symptom severity in host plants.

The genomic sequence of Wisteria vein mosaic virus and its similarities with other potyviruses

Summary.The complete nucleotide sequence of a Beijing isolate of Wisteria vein mosaic virus was determined to be 9695 nucleotides in length excluding the poly(A) tail. Sequence analysis predicted a single large open reading frame of 9279 nucleotides potentially encodes a polyprotein of 3092 amino acids. Phylogenetic analysis based on the genomic and deduced amino acid sequences support the current status of Wisteria vein mosaic virus (WVMV) as a distinct virus of the genus Potyvirus and a member of the Bean common mosaic virus (BCMV) subgroup. Sequence comparisons of WVMV and other members of the BCMV subgroup showed that WVMV is most closely related to both soybean mosaic virus and watermelon mosaic virus.

Hibiscus chlorotic ringspot virus p27 and Its Isoforms Affect Symptom Expression and Potentiate Virus Movement in Kenaf (Hibiscus cannabinus L.)

Hibiscus chlorotic ringspot virus (HCRSV), a member of the genus Carmovirus, encodes p27 (27-kDa protein) and two other in-frame isoforms (p25 and p22.5) that are coterminal at the carboxyl end. Only p27, which initiates at the 2570CUG codon, was detected in transfected kenaf (Hibiscus cannabinus L.) protoplasts through fusion to a Flag tag at either its N or C terminus. Subcellular localization of a p27-green fluorescent fusion protein in kenaf epidermal cells showed that it was localized to membrane structures close to cell walls. To study the functions of these proteins, a number of start codon mutants and premature translation termination mutants were constructed. Phenotypic differences were observed between the wild-type virus and these mutants during infection. Infectivity assays on plants indicated that p27 is a determinant of symptom severity. Without p25, appearance of symptoms on systemically infected kenaf leaves was delayed by 4 to 8 days. In a timecourse analysis, Western blot assays revealed that the delay corresponded to retardation in virus systemic movement, which suggested that p25 is probably involved in virus systemic movement. Mutations disrupting expression of p22.5 did not affect symptoms or virus movement.

The genomic sequence and biological properties of Pennisetum mosaic virus, a novel monocot-infecting potyvirus

The complete nucleotide sequences of two isolates of Pennisetum mosaic virus (PenMV) were determined. The viral genome comprised 9,611 nucleotides (nt) excluding the 3′-terminal poly(A) sequence, with the capacity of encoding a single polyprotein of 3,065 amino acids. The large open reading frame is flanked by a 172-nt 5′-untranslated region (UTR) and a 244-nt 3′-UTR. Sequence comparisons and phylogenetic analyses of the complete genome and polyproteins suggest that PenMV is closely related to other monocot potyviruses such as Maize dwarf mosaic virus, Sorghum mosaic virus and Sugarcane mosaic virus (SCMV), and thus represents a distinct potyvirus within the SCMV subgroup. The host range of PenMV is limited to Gramineae, and the virus naturally infects maize, sorghum and some wild grasses, causing mosaic symptoms on the leaves. This virus could be transmitted by both mechanical inoculation and by at least four species of aphids.

The genomic sequence of a Chinese isolate of Squash mosaic virus with novel 5′ conserved ends

Squash mosaic virus (SqMV) is a member of the genus Comovirus in the family Comoviridae [1]. It is a seedborne and beetle-transmitted virus infecting most plants in the genera Cucurbita and Cucumis [2, 3]. Like other comoviruses, SqMV has a bipartite positive-strand RNA genome consisting of RNA1 and RNA2, which are separately encapsidated in isometric particles of 28 nm in diameter. The genomes contain a poly(A) tail at the 30-terminus and the genome-linked viral protein (VPg) attached to the 50-end. It has been reported frequently in North and South America and Japan. Isolates from different regions have been extensively characterized and based on agar double-diffusion serological tests and host reactions, those isolates had been classified into different serogroups and biotypes [4–6]. In addition to the incomplete full length sequences a Japanese isolate of SqMV (Genbank accession numbers AB054689 and AB054688), the genomic RNA2 of three isolates from the United State have been sequenced. In China, the disease caused by SqMV has been considered one of the major limiting factors for the production of cucurbits [7], but the genomic sequence of chinese isolate of SqMV, SqMV-CH, has not yet been determined. The complete genomic sequence of SqMV-CH was determined from different overlapped cDNA clones and 50 and 30 RACE results. The SqMV-CH RNA1 contains 5,867 nucleotides excluding the poly(A) sequence of unknown length at 30-end. The sequence begins with GAAGA UGGUUAUUAAA, where the underlined UAUUAAA is the consensus sequence found at the 50-termini of other comoviruses [8, 9]. The overall base composition of RNA1 is 26.9% A, 18.4% C, 24.0% G, and 30.7% U, which is similar to reported RNA1 sequence of the Japanese isolate Y-SqMV. Computer-assisted analysis of SqMV-CH RNA1 nucleotide sequence revealed the presence of a single long open reading frame (ORF) which begins at position 245 (AUG245) and terminates at 5,819 UAA and encodes a single polyprotein of 1,858 amino acids with a calculated molecular mass of 209.4 kDa. The polyprotein of SqMV-CH RNA1 ORF has 44–47% amino acid identity to that of other comoviruses, respectively, which could be divided into five putative proteins. The SqMV-CH RNA2 has 3,395 nucleotides excluding the poly(A) tail, which begins with GAAGAUGGUU AUUAAA as same as RNA1. The strong conservation of the 50-termini of RNA1 and RNA2 were also found in the genome of SqMV-CH. The bases composition, SqMV-CH RNA2 (26.7% A, 20.3% C, 21.9% G, 31.1% U) is similar to that of RNA1 and it encodes a single ORF, which was predicted to release the movement protein and the larger and smaller coat protein, according to the reported sequence of SqMV RNA2. The comparison of the deduced amino acid sequences of polyproteins encoded by RNA2 in The nucleotide sequences reported in this study have been submitted to the Genbank database as accession number EU421059 and EU421060.