Crowdsourced Identification of Potential Target Genes for CTV Induced Gene Silencing for Controlling the Citrus Greening Vector Diaphorina citri

Abstract

Citrus Greening or Huanglongbing (HLB) is a disease of citrus, causing high reduction in citrus production and is transmitted by the Asian citrus psyllid Diaphorina citri Kuwayama vectoring a phloem-limited bacterium Candidatus Liberibacter sp. We report research results using crowdsourcing challenge strategy identifying potential gene targets in D. citri to control the insect using RNA interference (RNAi). From 63 submitted sequences, 43 were selected and tested by feeding them to D. citri using artificial diet assays. After feeding on artificial diet, the three most effective dsRNAs causing 30% mortality above control silenced genes expressing iron-sulfur cluster subunit of the mitochondrial electron transport chain complex (Rieske), heme iron-binding terminal oxidase enzyme (Cytochrome P450) and tetrahydrobiopterin (BH4) pathway enzyme (Pterin 4α-Carbinolamine Dehydratase). These sequences were cloned into a citrus phloem-limited virus (Citrus tristeza virus, CTV T36) expressing dsRNA against these target genes in citrus. The use of a viral mediated “para-transgenic” citrus plant system caused higher mortality to adult D. citri than what was observed using artificial diet, reaching 100% when detached citrus leaves with the engineered CTV expressing dsRNA were fed to adult D. citri. Using this approach, a virus-induced gene silencing (VIGS) can be used to test future transgenic cultivars before genetically engineering citrus. RNA Seq analysis after feeding D. citri CTV-RIE on infected leaves identified transcriptionally modified genes located upstream and downstream of the targeted RIE gene. These genes were annotated showing that many are associated with the primary function of the Rieske gene that was targeted by VIGS.

Detection of citrus vein phloem degeneration disease (Candidatus Liberibacter asiaticum) in orange cv. Selayar, Citrus reticulata L.

“…DNA bands of 1160 bp were found in columns 4, 11, and 12, while the other columns were negative. This is in line with the statement of [3], that not all leaves on twigs show symptoms of attack Positive CVPD contains CLa bacteria. It can happen that the upper leaves are positive for bacteria while the lower leaves are negative [3], also stated that this finding shows that there is no need for the presence of a pathogen on the plant leaves to cause disease symptoms or in other words, pathogens that are on the leaves of other plants can cause symptoms to appear on the leaves next to them or above and below them.…”

“…CVPD causes damage to the vessels of plant tissues, especially in the leaves so the photosynthetic process in plants can be hampered even to the point where photosynthesis blocks occur (photosynthesis cannot occur) due to CVPD which damages plant vascular cells [19]. In this study, the Diaphoryna citri vector was not found either at the rootstock source location or in the nursery due to several factors such as the absence of host plants other than citrus which are favored by the vector, such as kemuning which does not grow around nurseries or mother gardens and has not yet entered the bud formation new period [3] reported that Diaphorina citri tends to prefer yellowing because it is a plant that is resistant to pruning and has young buds continuously. The existence of other host plants around citrus plantations needs to be considered because they can become a reservoir for Diaphorina citri [10].…”

“…Candidatus Liberobacter asiaticum lives in the phloem of citrus plants and they cause characteristic symptoms [3]. The spread and attack of CLa can be influenced by geographical location, from one area to another and the invasion of the disease into gardens is caused by infected plant material, especially through the use of shoots or buds of the eye patch [16].…”

“…This bacterium lives in the phloem of citrus plants and causes typical symptoms in the form of chlorosis or yellowing of the leaves, the color of the veins becomes dark green, the leaves thicker, stiffer and the size of the leaves and fruit become smaller. Infected shoots and through the insect vector Diaphorina citri [3,4]. Until the 1980s, orange plantations in Selayar Islands District were still declared CVPD-free, but in 1992 it was reported that citrus plants were suspected of being infected with CVPD [5] reported that there were 125 citrus trees in Batang Matasapo Village, Bontomatene District, and in Polebunging Village, Bontomarannu District that were attacked by CVPD, both mild and severe, but the transmitting insect, Diaphorina citri Kuway, had not been found.…”

RNA Interference in Insects: From a Natural Mechanism of Gene Expression Regulation to a Biotechnological Crop Protection Promise

“…Other approaches not requiring plant modification have been developed. Plants infected with modified viruses have been widely used for screening potential RNAi target genes [ 244 , 245 , 246 , 247 ], and similar viruses may also confer protection to plants against fungi and nematodes [ 248 , 249 ]. The virus acts as a dsRNA factory during its replication in the plant.…”