Biology and Genetics of Plant-Pathogen Interactions

Aedes mosquito vectors transmit many viruses of global health concern, including dengue, chikungunya and Zika. These vector-borne viral diseases have a limited number of treatment options, and vaccines vary in their effectiveness. Consequently, integrated vector management is a primary strategy for disease control. However, the increasing emergence and spread of insecticide resistance is threatening the efficacy of vector control methods. Identifying mutations associated with resistance in vector populations is important to monitor the occurrence and evolution of insecticide resistance and inform control strategies. Rapid and cost-effective genome sequencing approaches are urgently needed. Here we present an adaptable targeted amplicon approach for cost-effective implementation within next generation sequencing platforms. This approach can identify single nucleotide polymorphisms (SNPs) and small insertions and deletions (indels) in genes involved in insecticide resistance in Aedes aegypti mosquitoes. We designed and tested eleven amplicons, which included segments of the ace-1 (carbamate target), the Voltage-Gated Sodium Channel (vgsc; pyrethroids, DDT and organochlorines), and rdl (dieldrin) genes; thereby covering established knockdown resistance (kdr) mutations (e.g., S989P, I1011M/V, V1016G/I and F1534C), with the potential to identify novel ones. The amplicon assays were designed with internal barcodes, to facilitate multiplexing of large numbers of mosquitoes at low cost, and were sequenced using an Illumina platform. Our approach was evaluated on 152 Ae. aegypti mosquitoes collected in Cabo Verde, an archipelago with a history of arbovirus outbreaks. The amplicon sequence data revealed 146 SNPs, including four non-synonymous polymorphisms in the vgsc gene, one in ace-1 and the 296S rdl mutation previously associated with resistance to organochlorines. The 296S rdl mutation was identified in 98% of mosquitoes screened, consistent with the past use of an organochlorine compound (e.g., DDT). Overall, our work shows that targeted amplicon sequencing is a rapid, robust, and cost-effective tool that can be used to perform high throughput monitoring of insecticide resistance.

Abstract This preprint has been reviewed and recommended by Peer Community In Evolutionary Biology (https://doi.org/10.24072/pci.evolbiol.100052). The cost of resistance, or the fitness effect of resistance mutation in absence of the drug, is a very widepsread concept in evolutionary genetics and beyond. It has represented an important addition to the simplistic view that resistance mutations should solely be considered as beneficial mutations. Yet, this concept also entails a series of serious difficulties in its definition, interpretation and current usage. In many cases, it may be simpler, clearer, and more insightful to study, measure and analyze the fitness effects of mutations across environments and to better distinguish those effects from ‘pleiotropic effects’ of those mutations.

Transmission of plant viruses by phytophagous hemipteran insects encompasses complex interactions underlying a continuum of processes involved in virus acquisition, retention and inoculation combined with vector feeding behavior. Here, we investigated the effects of dietary pH on whitefly (Bemisia tabaci) feeding behavior and release of Lettuce infectious yellows virus (LIYV) virions retained in the vector's foregut. Electrical penetration graph analysis revealed that variables associated with whitefly probing and ingestion did not differ significantly in pH (4, 7.4, and 9) adjusted artificial diets. To investigate virus retention and release, whiteflies allowed to acquire LIYV virions in a pH 7.4 artificial diet were fed pH 4, 7.4, or 9 virion-free artificial (clearing) diets. Immunofluorescent localization analyses indicated that virions remained bound to the foreguts of approximately 20%-24% of vectors after they fed on each of the 3 pH-adjusted clearing diets. When RNA preparations from the clearing diets were analyzed by reverse transcription (RT) nested-PCR and, in some cases, real-time qPCR, successful amplification of LIYV-specific sequence was infrequent but consistently repeatable for the pH 7.4 diet but never observed for the pH 4 and 9 diets, suggesting a weak pH-dependent effect for virion release. Viruliferous vectors that fed on each of the 3 pH-adjusted clearing diets transmitted LIYV to virus-free plants. These results suggest that changes in pH values alone in artificial diet do not result in observable changes in whitefly feeding behaviors, an observation that marks a first in the feeding of artificial diet by whitefly vectors; and that there is a potential causal and contingent relationship between the pH in artificial diet and the release/inoculation of foregut bound virions.

Autophagy plays a paramount role in mammalian antiviral immunity including direct targeting of viruses and their individual components, and many viruses have evolved measures to antagonize or even exploit autophagy mechanisms for the benefit of infection. In plants, however, the functions of autophagy in host immunity and viral pathogenesis are poorly understood. In this study, we have identified both anti- and proviral roles of autophagy in the compatible interaction of cauliflower mosaic virus (CaMV), a double-stranded DNA pararetrovirus, with the model plant Arabidopsis thaliana We show that the autophagy cargo receptor NEIGHBOR OF BRCA1 (NBR1) targets nonassembled and virus particle-forming capsid proteins to mediate their autophagy-dependent degradation, thereby restricting the establishment of CaMV infection. Intriguingly, the CaMV-induced virus factory inclusions seem to protect against autophagic destruction by sequestering capsid proteins and coordinating particle assembly and storage. In addition, we found that virus-triggered autophagy prevents extensive senescence and tissue death of infected plants in a largely NBR1-independent manner. This survival function significantly extends the timespan of virus production, thereby increasing the chances for virus particle acquisition by aphid vectors and CaMV transmission. Together, our results provide evidence for the integration of selective autophagy into plant immunity against viruses and reveal potential viral strategies to evade and adapt autophagic processes for successful pathogenesis.

Sugarcane (interspecific hybrids of Saccharum species) is an economically important crop that provides 70% of raw table sugar production worldwide and contributes, in some countries, to bioethanol and electricity production. Leaf scald, caused by the bacterial plant pathogen Xanthomonas albilineans, is one of the major diseases of sugarcane. Dissemination of X. albilineans is mainly ensured by contaminated harvesting tools and infected stalk cuttings. However, some strains of this pathogen are transmitted by aerial means and are able to survive as epiphytes on the sugarcane phyllosphere before entering the leaves and causing disease. Here we present a protocol to estimate the capacity of attachment of X. albilineans to sugarcane leaves. Tissue-cultured sugarcane plantlets were immersed in a bacterial suspension of X. albilineans and leaf attachment of X. albilineans was determined by two methods: leaf imprinting (semi-quantitative method) and leaf washing/homogenization (quantitative method). These methods are important tools for evaluating pathogenicity of strains/mutants of the sugarcane leaf scald pathogen.

Breeding new interspecific banana hybrid varieties relies on the use of Musa acuminata and M. balbisiana parents. Unfortunately, infectious alleles of endogenous Banana streak virus (eBSV) sequences are present in the genome of Musa balbisiana genitors. Upon activation by biotic and abiotic stresses, these infectious eBSVs lead to spontaneous infections by several species of Banana streak virus in interspecific hybrids harboring both Musa acuminata and M. balbisiana genomes. Here we provide evidence that seedy M. balbisiana diploids display diverse eBSV allelic combinations and that some eBSVs differ structurally from those previously reported. We also show that segregation of infectious and non-infectious eBSV alleles can be achieved in seedy M. balbisiana diploids through self-pollination or chromosome doubling of haploid lines. We report on the successful breeding of M. balbisiana diploid genitors devoid of all infectious eBSV alleles following self-pollination and on the potential of breeding additional M. balbisiana diploid genitors free of infectious eBSVs by crossing parents displaying complementary eBSV patterns. Our work paves the way to the safe use of M. balbisiana genitors for breeding banana interspecific hybrid varieties with no risk of activation of infectious eBSVs.

Albicidin is a potent DNA gyrase inhibitor produced by the sugarcane pathogenic bacterium Xanthomonas albilineans. Here we report the elucidation of the hitherto unknown structure of albicidin, revealing a unique polyaromatic oligopeptide mainly composed of p-aminobenzoic acids. In vitro studies provide further insights into the biosynthetic machinery of albicidin. These findings will enable structural investigations on the inhibition mechanism of albicidin and its assessment as a highly effective antibacterial drug.

Adaptive evolution is currently accepted as playing a significant role in biological invasions. Adaptations relevant to invasions are typically thought to occur either recently within the introduced range, as an evolutionary response to novel selection regimes, or within the native range, because of long-term adaptation to the local environment. We propose that recent adaptation within the native range, in particular adaptations to human-altered habitat, could also contribute to the evolution of invasive populations. Populations adapted to human-altered habitats in the native range are likely to increase in abundance within areas frequented by humans and associated with human transport mechanisms, thus enhancing the likelihood of transport to a novel range. Given that habitats are altered by humans in similar ways worldwide, as evidenced by global environmental homogenization, propagules from populations adapted to human-altered habitats in the native range should perform well within similarly human-altered habitats in the novel range. We label this scenario ‘Anthropogenically Induced Adaptation to Invade’. We illustrate how it differs from other evolutionary processes that may occur during invasions, and how it can help explain accelerating rates of invasions.