Study of the interaction between sour orange and Citrus tristeza virus 

Citrus tristeza virus (CTV) is the causal agent of tristeza, a major disease of citrus that forced to rebuild many citrus industries using tristeza tolerant rootstock. This closterovirus, transmitted by propagation of infected buds and by aphids, is adapted to replicate in the phloem cells of some species within the Rutaceae family. CTV induces three distinct syndromes i) decline and death of citrus varieties propagated on sour orange rootstock , ii) stem pitting, stunting and low bearing of some citrus varieties independently of the rootsock, and iii) seedling yellows of sour orange, grapefruit and lemon plants. CTV has a single-stranded, positive sense genomic RNA containing 12 open reading frames, potentially encoding at least 17 proteins, and two untranslated 3´ and 5´ terminal regions. Three of these proteins (p25, p20 and p23) act as suppressors of RNA silencing in Nicotiana benthamiana y N. tabacum plants. Although the pathogenicity determinants are presently unknown, experiments with transgenic plants expressing p23 indicated that this protein is involved in symptom expression. It is also unknown if symptom intensity is related to viral accumulation or to other factors.  
To assess if symptom differences between CTV isolates or between hosts infected with the same isolate were related with viral accumulation Mexican lime (ML), Citrus macrophylla (CM), sweet orange (SwO) and sour orange (SO) plants were inoculated with four isolates, inducing different symptom intensity in these hosts. Viral accumulation was estimated by ELISA and quantitative real time RT-PCR at 3 and 9 months post inoculation. Differences in viral load between hosts and isolates were not related with virulence, suggesting that the CTV symptom intensity is not a direct result of viral accumulation. Moreover, the four isolates showed an accumulation pattern in SO different from that observed in the other hosts, suggesting that SO displays an initial resistance to CTV invasion. 
To examine if differences in the accumulation patterns shown by CTV isolates were related to variations in viral replication or in RNA-mediated gene silencing, the accumulation of subgenomic RNAs expressing the p20 and p23 proteins, and of CTV-derived siRNAs during the viral infection were analyzed using the same host/isolate combinations. Kinetics of CTV replication paralelled that of viral accumulation. In CTV susceptible hosts (ML, CM and SwO) the highest viral replication and the maximum viral accumulation were detected in the first flush after inoculation, whereas in SO both viral replication and viral accumulation increased during the course of the infection. Likewise, while in ML, CM and SwO the CTV-derived siRNAs were detected in first flush after inoculation, activation of silencing in SO occurred at a later stage. These results indicated that the initial resistance of SO to CTV accumulation was not related to RNA silencing, but rather it probably resulted from an inefficient interaction between viral and host factors needed for CTV movement in the plant. Differences in viral accumulation or silencing patterns shown by CTV isolates in distinct hosts, were neither associated with the suppressor activity of their p23 proteins, as revealed by transitory expression experiments in N. benthamiana plants. 
The limitation to CTV systemic invasion in SO was analyzed by examining the viral distribution of CTV isolates in SO plants, and the development of viral and CTV-derived siRNA accumulation patterns in the roots and canopy of SO seedlings or plants propagated on Carrizo citrange (resistant to CTV) or SwO (susceptible to CTV) rootstocks. SO exhibited a limitation of cell to cell and long distance movement, which delayed systemic invasion and viral accumulation at the canopy, with variable patterns depending on the isolate and the rootstock. The virus was initially detected in the roots and its accumulation level affected the viral load in the canopy depending on the CTV isolate. Accumulation of the isolate T385 in the canopy was associated to the viral accumulation in roots, suggesting that these were acting as a viral reservoir for this genotype. 
Finally, cDNA microarray technology was used to analyze at the transcriptomic level the response of SO to CTV at the early stages of infection. The results confirmed our previous hypothesis that i) in SO there was a delay in the activation of  plant defense mechanisms probably due to delayed viral accumulation, and ii) this delay  likely results from a limitation in the cell to cell movement. 

SUMMARY

SUMMARY