Isolation of novel citrus and plum fruit promoters and their functional characterization for fruit biotechnology.

BACKGROUND: Promoters that confer expression in fruit tissues are important tools for genetic engineering of fruit quality traits, yet few fruit-specific promoters have been identified, particularly for citrus fruit development. RESULTS: In this study, we report five citrus fruit-specific/preferential promoters for genetic engineering. Additionally, we have characterized a novel fruit-preferential promoter from plum. Genes specifically expressed in fruit tissues were selected and their isolated promoter regions were fused with the GUSPlus reporter gene for evaluation in transgenic plants. Stable transformation in Micro-Tom tomato demonstrated that the candidate promoter regions exhibit differing levels of expression and with varying degrees of fruit specificity. CONCLUSIONS: Among the five candidate citrus promoters characterized in this study, the CitSEP promoter showed a fruit-specific expression pattern, while the CitWAX and CitJuSac promoters exhibited high fruit-preferential expression with strong activity in the fruit, weak activity in floral tissues and low or undetectable activity in other tissues. The CitVO1, CitUNK and PamMybA promoters, while exhibiting strong fruit-preferential expression, also showed consistent weak but detectable activity in leaves and other vegetative tissues. Use of these fruit specific/preferential promoters for genetic engineering can help with precise expression of beneficial genes and help with accurate prediction of the activity of new genes in host fruit plants.

Dynamic changes impact the plum pox virus population structure during leaf and bud development.

Plum pox virus (PPV) is a worldwide threat to stone fruit production. Its woody perennial hosts provide a dynamic environment for virus evolution over multiple growing seasons. To investigate the impact seasonal host development plays in PPV population structure, next generation sequencing of ribosome associated viral genomes, termed translatome, was used to assess PPV variants derived from phloem or whole leaf tissues over a range of plum leaf and bud developmental stages. Results show that translatome PPV variants occur at proportionately higher levels in bud and newly developing leaf tissues that have low infection levels while more mature tissues with high infection levels display proportionately lower numbers of viral variants. Additional variant analysis identified distinct groups based on population frequency as well as sets of phloem and whole tissue specific variants. Combined, these results indicate PPV population dynamics are impacted by the tissue type and developmental stage of their host.

Translatome Profiling of Plum Pox Virus-Infected Leaves in European Plum Reveals Temporal and Spatial Coordination of Defense Responses in Phloem Tissues.

Plum pox virus (PPV) is the causative agent of sharka, a devastating disease of stone fruits including peaches, apricots, and plums. PPV infection levels and associated disease symptoms can vary greatly, depending upon the virus strain, host species, or cultivar as well as developmental age of the infected tissues. For example, peaches often exhibit mild symptoms in leaves and fruit while European plums typically display severe chlorotic rings. Systemic virus spread into all host tissues occurs via the phloem, a process that is poorly understood in perennial plant species that undergo a period of dormancy and must annually renew phloem tissues. Currently, little is known about how phloem tissues respond to virus infection. Here, we used translating ribosome affinity purification followed by RNA sequencing to identify phloem- and nonphloem-specific gene responses to PPV infection during leaf development in European plum (Prunus domestica L.). Results showed that, during secondary leaf morphogenesis (4- and 6-week-old leaves), the phloem had a disproportionate response to PPV infection with two- to sixfold more differentially expressed genes (DEGs) in phloem than nonphloem tissues, despite similar levels of viral transcripts. In contrast, in mature 12-week-old leaves, virus transcript levels dropped significantly in phloem tissues but not in nonphloem tissues. This drop in virus transcripts correlated with an 18-fold drop in phloem-specific DEGs. Furthermore, genes associated with defense responses including RNA silencing were spatially coordinated in response to PPV accumulation and were specifically induced in phloem tissues at 4 to 6 weeks. Combined, these findings highlight the temporal and spatial dynamics of leaf tissue responses to virus infection and reveal the importance of phloem responses within a perennial host.