[Intellectual contribution]

Identification of ARL8, a host protein that is required for tomato mosaic virus multiplication

Masaki Nishikiori1, Hideyasu Okamura2, Hongyu Xiang2, Masashi Mori3, Koji Dohi3, Etsuko Katoh2, Satoshi Naito4, Tetsuo Meshi5, Masayuki Ishikawa1
1Plant-Microbe Interactions Research Unit, NIAS, 2Biomolecular Research Unit, NIAS, 3Ishikawa Prefectural University; and 4Hokkaido University, 5Division of Plant Sciences, NIAS
［Abstract］
We identified a host protein that co-purified with the replication proteins of Tomato mosaic virus (ToMV) as ARL8. Disruption of the ARL8 genes in plants completely inhibited ToMV multiplication without affecting plant growth. ARL8 played a role in enzymatic activation of ToMV replication proteins. In addition, we revealed the three-dimensional solution structures of ARL8 by NMR spectrometry.
［Keywords］
plant virus, replication, host factor, NMR, solution structure

［Background］

Tomato mosaic virus (ToMV) and other related viruses (collectively called ‘Tobamoviruses’) infect tomato and many other crop plants and reduce the yield and quality of agricultural products. Virions of Tobamoviruses are stable and remain active in soil for many years. While strong and durable resistance genes to Tombamoviruses are known for some crops, such genes are not available for others. In this study, we aimed to identify host factors required for tobamovirus multiplication to establish new strategies to control infection of the viruses.

［Results and Discussion］
  1. We identified a protein that co-purified with ToMV replication proteins from infected plant cells as ARL8 by determining partial amino acid sequences. ARL8 is a small GTPbinding protein belonging to the ARF/ARL family, whose members are known to be involved in membrane trafficking.
  2. We established lines of Arabidopsis thaliana and Nicotiana tabacum in which three ARL8 genes were disrupted. These plants grew normally, but were completely resistant to ToMV and Tobacco mosaic virus (TMV)-Cg, another Tobamovirus (Fig. 1). Cucumber mosaic virus (CMV), which belongs to a genus other than Tobamovirus, multiplied normally in these plants.
  3. Like ARL8, the host protein TOM1 is necessary for ToMV multiplication. We found that ToMV replication proteins, TOM1 and ARL8 interact with each other. ToMV replication proteins gained guanylyltransferase activity when TOM1 and ARL8 were co-expressed. These results suggest that TOM1 and ARL8 are components of the ToMV replication complex and play roles in enzymatic activation of the replication proteins (Fig. 2).
  4. ARF/ARL family proteins bind GTP or GDP and function as molecular switches. We determined the three-dimensional solution structures of the GTP-bound and GDPbound forms of ARL8, and revealed the mechanism of structural interconversion (Fig. 3). This structural interconversion may be important for ToMV RNA replication.
［Future prospects］
  1. It is expected that resistance to Tobamoviruses can be introduced to a variety of plants by suppressing ARL8 expression.
  2. The solution structures and mechanism of structural interconversion have been uncovered for ARL8. We plan to utilize this information to develop anti-tobamovirus substances that inhibit the conformational interconversion in ARL8 or the interaction between ARL8 and ToMV replication proteins.
Fig. 1. Analysis of A. thaliana arl8 mutants (A) Morphology of an arl8 triple mutant (right) compared with the wild type (left). (B) Multiplication of TMV-Cg and CMV in arl8 mutants. A. thaliana has three ARL8 genes. TMV-Cg or CMV RNAs were inoculated onto A. thaliana plants that lack either one (a, b, c), two (ac, bc, ab) or three (abc) ARL8 genes. Total proteins were prepared from the inoculated leaves two days after inoculation, and analyzed by SDSPAGE and immunoblottiong to detect the coat proteins. The accumulation of TMV-Cg coat protein was specifically inhibited in the mutants ab and abc.

Fig. 1. Analysis of A. thaliana arl8 mutants
(A) Morphology of an arl8 triple mutant (right) compared with the wild type (left). (B) Multiplication of TMV-Cg and CMV in arl8 mutants. A. thaliana has three ARL8 genes. TMV-Cg or CMV RNAs were inoculated onto A. thaliana plants that lack either one (a, b, c), two (ac, bc, ab) or three (abc) ARL8 genes. Total proteins were prepared from the inoculated leaves two days after inoculation, and analyzed by SDSPAGE and immunoblottiong to detect the coat proteins. The accumulation of TMV-Cg coat protein was specifically inhibited in the mutants ab and abc.


Fig. 2. The role of ARL8 and TOM1 in ToMV RNA replication ToMV replication protein is produced in an inactive form, and acquires enzymatic activity necessary for viral RNA replication through interaction with TOM1 and ARL8.

Fig. 2. The role of ARL8 and TOM1 in ToMV RNA replication ToMV replication protein is produced in an inactive form, and acquires enzymatic activity necessary for viral RNA replication through interaction with TOM1 and ARL8.
Fig. 3. Solution structures of the ARL8 protein. The structure was determined by using NMR spectrometry. When Mg2+ concentration is low, GDPbound ARL8 mainly takes so-called GDP form (right). In this condition, only a low population GDP-bound ARL8 takes the GTP-like form (left). On the other hand, in the presence of Mg2+, the GTP-like conformation is stabilized and a higher population of GDP-bound ARL8 adopts this conformation.

Fig. 3. Solution structures of the ARL8 protein. The structure was determined by using NMR spectrometry. When Mg2+ concentration is low, GDPbound ARL8 mainly takes so-called GDP form (right). In this condition, only a low population GDP-bound ARL8 takes the GTP-like form (left). On the other hand, in the presence of Mg2+, the GTP-like conformation is stabilized and a higher population of GDP-bound ARL8 adopts this conformation.

[Reference]

  1. Nishikiori M, Mori M, Dohi K, Okamura H, Katoh E, Naito S, Meshi T and Ishikawa M (2011) A host small GTP-binding protein ARL8 plays crucial roles in tobamovirus RNA replication PLoS Pathogens 7(12):e1002409
  2. Okamura H, Nishikiori M, Xiang H, Ishikawa M and Katoh E (2011) Interconversion of two GDP-bound conformations and their selection in an Arf-family small G protein Structure 19(7):988-998
  3. Patent application# JP-2007-14197 (Japan)
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