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Tomohide Natsuaki
Faculty of Agriculture, Utsunomiya University
Mine-machi 350, Utsunomiya 321-8505, Japan


Cross protection is a phenomenon whereby prior infection with one virus (the primary virus) prevents or interferes with subsequent infection by another isolate of the same virus or a closely related virus (the secondary virus). This suggests that cross protection using attenuated viruses appears to offer a promising strategy for biological control of plants viral diseases. A few attenuated viruses have been commercially used as a "vaccine" in Japan. Because cross protection is effective in general, it is necessary to increase good attenuated strains against many severe viruses. To study the underlying molecular mechanism, it is essential to know how the attenuated isolate differs in genome structure from the original virulent isolate.

Key words:     attenuation, mild strain, cross protection, Cucumber mosaic virus, satellite RNA, 2b protein, potyvirus, HC-Pro


Tomato mosaic virus (ToMV) was once one of the most important pathogens of tomato (Solanum lycopersicum) plants, but effective control has been established using resistant cultivars with three ToMV resistance genes. However, resistant varieties of tomato to Cucumber mosaic virus (CMV) are not yet commercially available. In the 1970s and the 1980s, CMV caused serious damage to processing tomato plants in the main production areas of Tochigi and Nagano prefectures, Japan. In these areas, a large number of farmers stopped growing processing tomatoes. At that time CMV was one of the most important emerging viruses and greatly influenced some Japanese researchers to realize the importance of reliable and rapid methods of virus detection and diagnosis. Because control of plant viruses must be largely preventive, the collaboration of researchers is very important to develop mild strains (vaccine) against emerging viruses like CMV and to study the molecular mechanisms of viral attenuation and cross protection.

Cross protection is a phenomenon whereby prior infection with one virus (the primary virus) prevents or interferes with subsequent infection by another isolate of the same virus or a closely related virus (the secondary virus). The phenomenon was first reported with Tobacco mosaic virus (TMV) in 1929. Since then, cross protection has been demonstrated for many plant viruses including sap-transmissible viruses such as Potato virus X (PVX), non-sap-transmissible Potato leaf roll virus (PLRV) and Citrus tristeza virus (CTV), RNA viruses, DNA viruses, and viroids (Gal-On and Shiboleth 2006; Pennazio et al. 2001).

Cross protection appears to offer a promising strategy for biological control of plant viruses. This method has been used to control virus diseases in several crops worldwide. However, the selection and production of mild strains for this control strategy has been dependent upon various empirical methods based on symptomatology, back-inoculation to indicator plants, or both; but these methods are time-consuming and, without appropriate indicator plants, often inconclusive. To date, the mechanism of cross protection is not yet clearly understood. To study the underlying molecular mechanism, it is essential to know how the attenuated isolate differs in genome structure from the original virulent isolate.

Cucumber mosaic virus

Because CMV has a wide host range and can be transmitted by more than 80 species of aphids, CMV outbreaks are difficult to control. For CMV, cross protection was obtained using two types of mild strains, either with or without a satellite RNA of the ameliorating phenotype.

Sayama et al. (1993) isolated a potentially useful, attenuated CMV that had an associated nonnecrogenic satellite RNA with microsequence heterogeneity. The isolate caused no symptoms or very mild symptoms in general on infected plants and has been used in commercial fields in Japan until now. We also showed that multimers of the double-stranded and the plus-sense of single-strand satellite RNAs are present in CMV-infected plants, but that the minus-sense of the single-strand form is absent (Kuroda et al. 1997).

Various satellite RNAs were isolated to effect a change in symptoms induced on host plants by CMV, while other satellite RNAs may have no effect on viral symptoms (Natsuaki et al. 1994; Wang et al. 1990). In combination with a given strain of CMV, different satellite RNAs can attenuate viral symptoms, induce chlorosis, induce necrosis, or induce combinations of these effects. We also conjectured that genetic reassortment of CMV RNA may occur in nature following mixed infection and be an important mechanism for generating CMV variation (Kuroda et al. 2005; Wang et al. 1988). Since RNA silencing in plants must be playing an important role in the pathogenicity of CMV satellites RNA, further molecular study is necessary regarding the effects of satellite RNA on the pathogenicity of CMV.

On the other hand, strain CM95, selected as a good attenuated CMV isolate, produces no or mild symptoms on inoculated plants and confers strong cross protection to plants against infection by severe isolates. We showed that the attenuation by CM95 originated in a single amino acid mutation in 2b protein, a known RNA silencing suppressor. The 2b protein of CM95 is weakly suppressive in comparison with those of other severe CMV isolates (Nakazono-Nagaoka et al. 2005; Natsuaki et al. 2008). In addition, we showed that the weak suppressor activity of CM95 resulted from a loss of the siRNA-binding property of 2b via a single amino acid change (Goto et al. 2007).


Potyviruses form the largest group of plant viruses and infect many crops, causing economically important diseases around the world. Zucchini yellow mosaic virus (ZYMV) is a species of the genus Potyvirus, and strain ZYMV-2002 has been developed as a practically useful attenuated isolate (Kosaka et al. 2007; Wang et al. 2006). Comparison of the complete nucleotide sequences of ZYMV-2002 and the parental severe isolate Z5-1 revealed 14 nucleotide differences, which resulted in seven amino acid changes (Wang et al. 2006). Full-length infectious cDNA clones of parental Z5-1 and attenuated ZYMV-2002 isolates were constructed to identify which amino acid changes are concerned with reduced symptom expression, and four amino acid mutations in the helper component proteinase (HC-Pro) region were clearly found to contribute to the attenuated symptoms of ZYMV-2002 (Kosaka et al. 2007).

The HC-Pro protein of potyviruses is well known to function in proteolytic activity, aphid transmission, viral long-distance movement in infected plants, virulence, maintenance of viral genome amplification, and suppression of host-defense RNA silencing. A unique amino acid substitution in the conserved FRNK motif of HC-Pro dramatically reduced symptom expression in cucurbit plants infected with ZYMV (Gal-On and Shiboleth 2006). However, the amino acid mutations in the HC-Pro region of ZYMV-2002 were not located in the FRNK motif. The mutations in ZYMV-2002 were suggested to be responsible for the lack of aphid transmissibility and for producing invisible local lesions on Chenopodium quinoa (Wang et al. 2006). Similar to the mutation in HC-Pro of Bean yellow mosaic virus (BYMV), a member of potyviruses, the mutation might have altered the protein's suppressive activity and be associated with the attenuation (Nagaoka-Nakazono et al. 2004, 2009).

Improvement of selection and identification of attenuated viruses

It is usually impossible to discern attenuated strains from severe ones by serological methods. Therefore, molecular analysis of attenuated and severe viruses is important for assessing the protective ability of attenuated strains (vaccine) in the field. Reverse transcription-polymerase chain reaction (RT-PCR) or RT-PCR-restriction fragment length polymorphism techniques were developed to rapidly differentiate mild strains from severe ones by comparing their genome sequences (Nakazono-Nagaoka et al. 2004, 2005).

The process of selecting stable attenuated strains with strong cross-protection ability is time-consuming and is a major obstacle to our progress in using mild strains to control viral diseases. Thus, an efficient technique to select a good attenuated virus to control CMV disease had to be developed. In our study, mild CMV isolates appeared to be more efficiently selected from dark-green leaf tissue, suggesting that isolations from dark-green tissues after mutagenesis may be advantageous for several plant viruses (Kobori et al. 2005).

Molecular mechanisms of cross protection

Several hypotheses have been proposed to explain how infection with a primary virus can prevent infection with a second virus: (i) the formation of "antibodies" in plants, which would prevent second virus infection, (ii) the encapsidation of the second strain RNA by the coat protein (CP) of the primary strain or the prevention of encapsidation of the second strain RNA by the CP of the primary strain, (iii) competition between strains for components essential for viral replication, (iv) the occupation of replication sites by the primary strain, and so on (Gal-On and Shiboleth 2006; Pennazio et al. 2001). However, only the CP-mediated and RNA-mediated cross protection explanations are widely accepted.

The CP-mediated cross-protection model is based on the finding that transgenic plants expressing TMV-CP are resistant to TMV infection, suggesting that the uncoating of the second strain was prevented (Beachy 1999). However, this is not the only potential mechanism of cross protection because CP-defective viruses and viroids can confer cross protection. Another model, based on RNA silencing, was thus suggested and accepted to explain the cross-protection phenomenon for RNA and DNA viruses, as well as for viroids (Gal-On and Shiboleth 2006).


In our research, we inserted different fluorescent genes (CFP or YFP) into infectious clones of attenuated and severe isolates to study the distribution of different populations of strains in mixed-infected plants. The results indicated that identical but differently labeled strain populations were always distributed separately in coinfected leaves (Natsuaki 2009; Takahashi et al. 2007).

Plasmids containing the same origin of replication are generally considered incompatible (Nordström 1993; Novick 1987). In other words, if the introduction of a second plasmid destabilizes the inheritance of the first, the two are said to be incompatible. It is suggested that incompatibility is due to the sharing of one or more elements of the plasmid replication or partitioning systems. I feel there is some similarity between plasmid incompatibility and cross protection. And the resistance in transgenic plants that is conferred by the introduction of a virus gene is lost upon infection by unrelated viruses but the cross protection is not. From various viewpoints, the molecular mechanism underlying cross protection between plant viruses remains a mystery.

A few attenuated viruses have been commercially used as a "vaccine". Because cross protection is effective in general, it is necessary to increase good attenuated strains against many severe viruses. Based on the molecular research of attenuation and cross protection, new techniques might be developed to further our progress in controlling viral diseases through cross protection with attenuated viruses.


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