Superinfection exclusion, a phenomenon in which a preexisting viral infection prevents a secondary infection with the same or a closely related virus, has been described for various viruses, including important pathogens of humans, animals, and plants. The phenomenon was initially used to test the relatedness of plant viruses. Subsequently, purposeful infection with a mild isolate has been implemented as a protective measure against virus isolates that cause severe disease. In the medical and veterinary fields, superinfection exclusion was found to interfere with repeated applications of virus-based vaccines to individuals with persistent infections and with the introduction of multicomponent vaccines. In spite of its significance, our understanding of this phenomenon is surprisingly incomplete. Recently, it was demonstrated that superinfection exclusion of Citrus tristeza virus (CTV), a positive-sense RNA closterovirus, occurs only between isolates of the same strain, but not between isolates of different strains of the virus. In this study, I show that superinfection exclusion by CTV requires production of a specific viral protein, the p33 protein. Lack of the functional p33 protein completely eliminated the ability of the virus to exclude superinfection by the same or a closely related virus. Remarkably, the protein appeared to function only in a homology-dependent manner. A cognate protein from a heterologous strain failed to confer the exclusion, suggesting the existence of precise interactions of the p33 protein with other factors involved in this complex phenomenon.

Citrus greening or Huanglongbing (HLB) is caused by the phloem-limited intracellular Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). HLB-infected citrus phloem cells undergo structural modifications that include cell wall thickening, callose and phloem protein induction, and cellular plugging. However, very little is known about the intracellular mechanisms that take place during CLas cell-to-cell movement. Here, we show that CLas movement through phloem pores of sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi) is carried out by the elongated form of the bacteria. The round form of CLas is too large to move, but can change its morphology to enable its movement. CLas cells adhere to the plasma membrane of the phloem cells specifically adjacent to the sieve pores. Remarkably, CLas was present in both mature sieve element cells and nucleated nonsieve element cells. The sieve plate plugging structures of host plants were shown to have different composition in different citrus tissues. Callose deposition was the main plugging mechanism in the HLB-infected flush, where it reduced the open space of the pores. In the roots, pores were surrounded by dark extracellular material, with very little accumulation of callose. The expression of CALLOSE SYNTHASE7 and PHLOEM PROTEIN2 genes was upregulated in the shoots, but downregulated in root tissues. In seed coats, no phloem occlusion was observed, and CLas accumulated to high levels. Our results provide insight into the cellular mechanisms of Gram-negative bacterial cell-to-cell movement in plant phloem.

Citrus tristeza virus (CTV) causes two citrus diseases that have caused devastating losses in global citrus production. The first disease is quick decline of trees propagated on the sour orange rootstock. The second disease is stem pitting, which severely affects a number of economically important citrus varieties regardless of the rootstock used and results in reduced tree growth and vigor as well as in reduced fruit size and quality. Both diseases continue to invade new areas. While quick decline could be effectively managed by the use of resistant and/or tolerant rootstocks, the only means to protect commercial citrus against endemic stem pitting isolates of CTV has been cross-protection with mild isolates of the virus. In some citrus areas cross-protection has been successful and allowed production of certain citrus cultivars despite the presence of severe stem pitting isolates in those regions. However, many other attempts to find isolates that would provide sustained protection against aggressive isolates of the virus had failed. In general, there has been no understanding why some mild isolates were effective and others failed to protect. We have been working on the mechanism of cross-protection by CTV. Recent considerable progress has significantly advanced our understanding of how cross-protection may work in the citrus/CTV pathosystem. As we demonstrated, only isolates that belong to the same strain of the virus cross protect against each other, while isolates from different strains do not. We believe that the results of our research could now make finding protecting isolates relatively straightforward. This review discusses some of the history of CTV cross-protection along with the recent findings and our ‘recipe’ for selection of protecting isolates.

Citrus tristeza virus (CTV) is a phloem-limited virus whose natural host range is restricted to citrus and related species. Although the virus has killed millions of trees, almost destroying whole industries, and continually limits production in many citrus growing areas, most isolates are mild or symptomless in most of their host range. There is little understanding of how the virus causes severe disease in some citrus and none in others. Movement and distribution of CTV differs considerably from that of well-studied viruses of herbaceous plants where movement occurs largely through adjacent cells. In contrast, CTV systemically infects plants mainly by long-distance movement with only limited cell-to-cell movement. The virus is transported through sieve elements and occasionally enters an adjacent companion or phloem parenchyma cell where virus replication occurs. In some plants this is followed by cell-to-cell movement into only a small cluster of adjacent cells, while in others there is no cell-to-cell movement. Different proportions of cells adjacent to sieve elements become infected in different plant species. This appears to be related to how well viral gene products interact with specific hosts. CTV has three genes (p33, p18, and p13) that are not necessary for infection of most of its hosts, but are needed in different combinations for infection of certain citrus species. These genes apparently were acquired by the virus to extend its host range. Some specific viral gene products have been implicated in symptom induction. Remarkably, the deletion of these genes from the virus genome can induce large increases in stem pitting (SP) symptoms. The p23 gene, which is a suppressor of RNA silencing and a regulator of viral RNA synthesis, has been shown to be the cause of seedling yellows (SY) symptoms in sour orange. Most isolates of CTV in nature are populations of different strains of CTV. The next frontier of CTV biology is the understanding how the virus variants in those mixtures interact with each other and cause diseases.

Superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by the same or a closely related virus, has been described for different viruses, including important pathogens of humans, animals, and plants. Citrus tristeza virus (CTV), a positive-sense RNA virus, represents a valuable model system for studying SIE due to the existence of several phylogenetically distinct strains. Furthermore, CTV allows SIE to be examined at the whole-organism level. Previously, we demonstrated that SIE by CTV is a virus-controlled function that requires the viral protein p33. In this study, we show that p33 mediates SIE at the whole-organism level, while it is not required for exclusion at the cellular level. Primary infection of a host with a fluorescent protein-tagged CTV variant lacking p33 did not interfere with the establishment of a secondary infection by the same virus labeled with a different fluorescent protein. However, cellular coinfection by both viruses was rare. The obtained observations, along with estimates of the cellular multiplicity of infection (MOI) and MOI model selection, suggested that low levels of cellular coinfection appear to be best explained by exclusion at the cellular level. Based on these results, we propose that SIE by CTV is operated at two levels—the cellular and the whole-organism levels—by two distinct mechanisms that could function independently. This novel aspect of viral SIE highlights the intriguing complexity of this phenomenon, further understanding of which may open up new avenues to manage virus diseases. IMPORTANCE Many viruses exhibit superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by related viruses. SIE plays an important role in the pathogenesis and evolution of virus populations. The observations described here suggest that SIE could be controlled independently at different levels of the host: the whole-organism level or the level of individual cells. The p33 protein of citrus tristeza virus (CTV), an RNA virus, was shown to mediate SIE at the whole-organism level, while it appeared not to be required for exclusion at the cellular level. SIE by CTV is, therefore, highly complex and appears to use mechanisms different from those proposed for other viruses. A better understanding of this phenomenon may lead to the development of new strategies for controlling viral diseases in human populations and agroecosystems.