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Combinations of two amino acids (Ala⁴⁰ and Phe⁷⁵ or Ser⁴⁰ and Tyr⁷⁵) in the coat protein of apple chlorotic leaf spot virus are crucial for infectivity

¹ The United Graduate School of Agricultural Sciences, Iwate University, Morioka 020-8550, Japan
² Plant Pathology Laboratory, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan
³ Plant Pathology Laboratory, Faculty of Agriculture and Life Sciences, Hirosaki University, Hirosaki 036-8561, Japan

Correspondence
Nobuyuki Yoshikawa
yoshikawa{at}iwate-u.ac.jp

Amino acid sequences of apple chlorotic leaf spot virus (ACLSV) coat protein (CP) were compared between 12 isolates from apple, plum and cherry, and 109 cDNA clones that were amplified directly from infected apple tissues. Phylogenetic analysis based on the amino acid sequences of CP showed that the isolates and cDNA clones were separated into two major clusters in which the combinations of the five amino acids at positions 40, 59, 75, 130 and 184 (Ala⁴⁰-Val⁵⁹-Phe⁷⁵-Ser¹³⁰-Met¹⁸⁴ or Ser⁴⁰-Leu⁵⁹-Tyr⁷⁵-Thr¹³⁰-Leu¹⁸⁴) were highly conserved within each cluster. Site-directed mutagenesis using an infectious cDNA clone of ACLSV indicated that the combinations of two amino acids (Ala⁴⁰ and Phe⁷⁵ or Ser⁴⁰ and Tyr⁷⁵) are necessary for infectivity to Chenopodium quinoa plants by mechanical inoculation. Moreover, an agroinoculation assay indicated that the substitution of a single amino acid (Ala⁴⁰ to Ser⁴⁰ or Phe⁷⁵ to Tyr⁷⁵) resulted in extreme reduction in the accumulation of viral genomic RNA, double-stranded RNAs and viral proteins (movement protein and CP) in infiltrated tissues, suggesting that the combinations of the two amino acids at positions 40 and 75 are important for effective replication in host plant cells.

The GenBank/EMBL/DDBJ accession numbers for the sequences obtained in this study are AB326223–AB326230.