Periodic Reporting for period 1 - GuardSym (Disentangling the multitrophic interactions of the supervector Bemisia tabaci to potentially use its symbiotic communities to reduce plants viral-vectored diseases.)
Période du rapport: 2021-02-01 au 2023-01-31
Agriculture is facing constant pressure to increase food production to fulfill human population demands. This has been accomplished by the use of intensive agricultural techniques. The increase in global temperatures together with intensive techniques like worldwide movements of plant material, use of monocultures, and abuse of insecticides have modified the trophic chains in agricultural systems. As a consequence, there is an unprecedented increase in emergent plant viral diseases in the last decades. Most of these viruses require an insect vector for their transmission. Unfortunately, some vector insects have been favored by intensive agriculture. Indeed, the whitefly Bemisia tabaci has become a worldwide supervector and is the cause of the global emergence of begomoviruses and criniviruses. This whitefly has developed resistance to many insecticides and developing new control techniques is a must.
Recently, a population modification strategy has been applied to mosquitoes, displacing natural populations and replacing them with virus-resistant ones that are no longer able to transmit human diseases. This success has been achieved using Wolbachia, a bacterial symbiont present in many insects, that can confer virus-resistance phenotypes to its host. Interestingly, B. tabaci presents a rich bacterial symbiotic community, including Wolbachia, that can be naturally manipulated through hybridization. This offers the unique opportunity to unravel the multitrophic interactions that occur between B. tabaci, its symbionts, the vectored virus, and the plant. Using integrative frameworks to study the insect and plant physiology, the virus transmission, the dynamics of the symbionts, and the cross-talks between them (their transcriptomes) can lead to the discovery of new symbiotic virus-resistant phenotypes in B. tabaci.
During the GuardSym action, two lines of B. tabaci carrying different symbiotic combinations but the same insect genetic background were generated. The Tomato Yellow Leaf Curly Virus (TYLCV) vectoring capabilities of the two lines obtained were compared to the parental and maternal lines. Whiteflies carrying TYLCV showed no differences in their survival or oviposition. Besides, the height, weight, and number of leaves of TYLCV symptomatic tomato plants were similar independently of the symbiotic community harbored by the insect. These results suggest that symbiotic communities induced TYLCV-resistance phenotypes neither in the insect host nor in the plant. Notwithstanding, it is possible that some resistance phenotypes could emerge in older plants or insects. Finally, both messenger RNA and small RNA were extracted from the two B. tabaci lines and tomato plants during the earlier and later stages of TYLCV disease. The molecular interactions between the insect, its symbionts, TYLCV, and the plant are currently under analysis.
Recently, a population modification strategy has been applied to mosquitoes, displacing natural populations and replacing them with virus-resistant ones that are no longer able to transmit human diseases. This success has been achieved using Wolbachia, a bacterial symbiont present in many insects, that can confer virus-resistance phenotypes to its host. Interestingly, B. tabaci presents a rich bacterial symbiotic community, including Wolbachia, that can be naturally manipulated through hybridization. This offers the unique opportunity to unravel the multitrophic interactions that occur between B. tabaci, its symbionts, the vectored virus, and the plant. Using integrative frameworks to study the insect and plant physiology, the virus transmission, the dynamics of the symbionts, and the cross-talks between them (their transcriptomes) can lead to the discovery of new symbiotic virus-resistant phenotypes in B. tabaci.
During the GuardSym action, two lines of B. tabaci carrying different symbiotic combinations but the same insect genetic background were generated. The Tomato Yellow Leaf Curly Virus (TYLCV) vectoring capabilities of the two lines obtained were compared to the parental and maternal lines. Whiteflies carrying TYLCV showed no differences in their survival or oviposition. Besides, the height, weight, and number of leaves of TYLCV symptomatic tomato plants were similar independently of the symbiotic community harbored by the insect. These results suggest that symbiotic communities induced TYLCV-resistance phenotypes neither in the insect host nor in the plant. Notwithstanding, it is possible that some resistance phenotypes could emerge in older plants or insects. Finally, both messenger RNA and small RNA were extracted from the two B. tabaci lines and tomato plants during the earlier and later stages of TYLCV disease. The molecular interactions between the insect, its symbionts, TYLCV, and the plant are currently under analysis.
The GuardSym action officially started on the 1st of February 2020 but the introgression of the Bemisia tabaci lines started during the second semester of 2019. However, during COVID-pandemic the lines were lost, forcing a re-start of the introgression process during the end of 2020 and 2021. By the beginning of 2022, two insect lines, with the same genetic background but different symbiotic compositions, were finally ready. Pending August to mid-September 2022, five B. tabaci lines, the two B. tabaci introgressed lines and their corresponding maternal (two) and paternal (one) lines, were transferred to the level 3 bio-security facilities (NS3) located at the Plant Health Institute of Montpellier (PHIM-CIRAD, France). All experiments were conducted inside the NS3 to avoid the escape of whiteflies infected by Tomato Yellow Leaf Curly Virus (TYLCV).
At the NS3, several experiments were performed to test if the different symbiotic communities of the insect vector interact with TYLCV and the host plant. For the first experiment, the fecundity of the five B. tabaci lines, previously infected with TYLCV, was recorded in two host plants: tomato (natural host of TYLCV) and cotton (non-host). In the second experiment, TYLCV-infected whiteflies were allowed to oviposit on cotton to test the ratio of TYLCV-infected eggs. Third, to measure the inoculation efficiency of the insect lines, TYLCV-infected insects were allowed to feed on cotton leaf discs for two days. The number of viral copies per leaf disc is being measured by quantitative PCR. The fourth experiment was to assess the TYLCV transmission efficiency of the different lines and the induction of resistance phenotypes in tomato plants. When introgressed lines (treatment) are compared between them or to their donor (control) lines, the data suggests that the symbiotic communities tested did not induce any resistance phenotype in the insect (lower fecundity or reduced TYLCV transmission compared to the donor lines). Similarly, when tomato plants infested with TYLCV-infected whitefly lines were compared, they had similar height, weight, and leave numbers independently of the symbiotic community harbored by the whitefly line. However, these results are partial and still need to be integrated with the ongoing analysis of TYLCV inoculation and vertical transmission and the molecular cross-talk between all the partners.
For that reason, a fifth experiment aimed to elucidate the molecular interactions between the insect, its symbionts, the virus, and the tomato host during the development of TYLC disease. For that, infected (treatment) and non-infected (control) whiteflies were allowed to feed on tomato plants. Then, total RNA was collected from whiteflies and plants at relevant time points during TYLC disease. Total RNA was used to obtain the messenger RNA (40 insect and 24 plant libraries) and small RNA (40 insect and 24 plant libraries) profiles. Differential expression and expression network analysis will be performed after the end of the action.
After the COVID-pandemic, the GuardSym action, and the preliminary results obtained, has been presented in different internal seminars (three), invited talks (four), general audience talk (one), international meetings (three). In addition, the action will be presented in future international meetings (e.g. Gordon Research Conference in Animal-Symbiosis 2023) and general audience events (e.g. Pint of Science Montpellier 2023 or The Week of Science Montpellier 2023). In addition, when all results will be ready and integrated, the Technology Transfer Office at the CNRS and the European IPR Helpdesk Exploitation will be contacted to discuss if any intellectual protection is required. Once clarified, the results of the action will be published in open-access journals.
At the NS3, several experiments were performed to test if the different symbiotic communities of the insect vector interact with TYLCV and the host plant. For the first experiment, the fecundity of the five B. tabaci lines, previously infected with TYLCV, was recorded in two host plants: tomato (natural host of TYLCV) and cotton (non-host). In the second experiment, TYLCV-infected whiteflies were allowed to oviposit on cotton to test the ratio of TYLCV-infected eggs. Third, to measure the inoculation efficiency of the insect lines, TYLCV-infected insects were allowed to feed on cotton leaf discs for two days. The number of viral copies per leaf disc is being measured by quantitative PCR. The fourth experiment was to assess the TYLCV transmission efficiency of the different lines and the induction of resistance phenotypes in tomato plants. When introgressed lines (treatment) are compared between them or to their donor (control) lines, the data suggests that the symbiotic communities tested did not induce any resistance phenotype in the insect (lower fecundity or reduced TYLCV transmission compared to the donor lines). Similarly, when tomato plants infested with TYLCV-infected whitefly lines were compared, they had similar height, weight, and leave numbers independently of the symbiotic community harbored by the whitefly line. However, these results are partial and still need to be integrated with the ongoing analysis of TYLCV inoculation and vertical transmission and the molecular cross-talk between all the partners.
For that reason, a fifth experiment aimed to elucidate the molecular interactions between the insect, its symbionts, the virus, and the tomato host during the development of TYLC disease. For that, infected (treatment) and non-infected (control) whiteflies were allowed to feed on tomato plants. Then, total RNA was collected from whiteflies and plants at relevant time points during TYLC disease. Total RNA was used to obtain the messenger RNA (40 insect and 24 plant libraries) and small RNA (40 insect and 24 plant libraries) profiles. Differential expression and expression network analysis will be performed after the end of the action.
After the COVID-pandemic, the GuardSym action, and the preliminary results obtained, has been presented in different internal seminars (three), invited talks (four), general audience talk (one), international meetings (three). In addition, the action will be presented in future international meetings (e.g. Gordon Research Conference in Animal-Symbiosis 2023) and general audience events (e.g. Pint of Science Montpellier 2023 or The Week of Science Montpellier 2023). In addition, when all results will be ready and integrated, the Technology Transfer Office at the CNRS and the European IPR Helpdesk Exploitation will be contacted to discuss if any intellectual protection is required. Once clarified, the results of the action will be published in open-access journals.
Nowadays, Bemisia tabaci is still considered one of the worst agricultural pest insects and continues to expand worldwide. Therefore, any increase on the basic knowledge on this insect will benefit our society. Unfortunately, the tested symbionts did not induce any observable resistance phenotype that might help to reduce the economical damage caused by this insect. The ongoing analysis of the molecular interactions happening in B. tabaci plant viral-vectored diseases can still allow the identification of target genes. Those genes can be used to develop new hypothesis and conduct basic research to understand their role in the disease. Also, they might help in the development of plant resistant cultivars.