Since the discovery of RNA interference (RNAi), scientists have made significant progress towards the development of this unique technology for crop protection. The RNAi mechanism works at the mRNA level by exploiting a sequence-dependent mode of action with high target specificity due to the design of complementary dsRNA molecules, allowing growers to target pests more precisely compared to conventional agrochemicals. The delivery of RNAi through transgenic plants is now a reality with some products currently in the market. Conversely, it is also expected that more RNA-based products reach the market as non-transformative alternatives. For instance, topically applied dsRNA/siRNA (SIGS – Spray Induced Gene Silencing) has attracted attention due to its feasibility and low cost compared to transgenic plants. Once on the leaf surface, dsRNAs can move directly to target pest cells (e.g., insects or pathogens) or can be taken up indirectly by plant cells to then be transferred into the pest cells. Water-soluble formulations containing pesticidal dsRNA provide alternatives, especially in some cases where plant transformation is not possible or takes years and cost millions to be developed (e.g., perennial crops). The ever-growing understanding of the RNAi mechanism and its limitations has allowed scientists to develop non-transgenic approaches such as trunk injection, soaking, and irrigation. While the technology has been considered promising for pest management, some issues such as RNAi efficiency, dsRNA degradation, environmental risk assessments, and resistance evolution still need to be addressed. Here, our main goal is to review some possible strategies for non-transgenic delivery systems, addressing important issues related to the use of this technology.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is one of the most important citrus pests. ACP is the vector of the phloem-limited bacteria Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus, the causal agents of the devastating citrus disease huanglongbing (HLB). The management of HLB is based on the use of healthy young plants, eradication of infected plants and chemical control of the vector. RNA interference (RNAi) has proven to be a promising tool to control pests and explore gene functions. Recently, studies have reported that target mRNA knockdown in many insects can be induced through feeding with double-stranded RNA (dsRNA). In the current study, we targeted the cathepsin D, chitin synthase and inhibitor of apoptosis genes of adult and nymph ACP by feeding artificial diets mixed with dsRNAs and Murraya paniculata leaves placed in dsRNAs solutions, respectively. Adult ACP mortality was positively correlated with the amount of dsRNA used. Both nymphs and adult ACP fed dsRNAs exhibited significantly increased mortality over time compared with that of the controls. Moreover, qRT-PCR analysis confirmed the dsRNA-mediated RNAi effects on target mRNAs. These results showed that RNAi can be a powerful tool for gene function studies in ACP and perhaps for HLB control.

Over the past few years, the use of RNA interference (RNAi) for insect pest management has attracted considerable interest in academia and industry as a pest-specific and environment-friendly strategy for pest control. For the success of this technique, the presence of core RNAi genes and a functional silencing machinery is essential. Therefore, the aim of this study was to test whether the Neotropical brown stinkbug Euschistus heros has the main RNAi core genes and whether the supply of dsRNA could generate an efficient gene silencing response. To do this, total mRNA of all developmental stages was sequenced on an Illumina platform, followed by a de novo assembly, gene annotation and RNAirelated gene identification. Once RNAi-related genes were identified, nuclease activities in hemolymph were investigated through an ex vivo assay. To test the functionality of the siRNA machinery, E. heros adults were microinjected with ~28 ng per mg of insect of a dsRNA targeting the V-ATPase-A gene. Mortality, relative transcript levels of V-ATPase-A, and the expression of the genes involved in the siRNA machinery, Dicer-2 (DCR-2) and Argonaute 2 (AGO-2), were analyzed. Transcriptome sequencing generated more than 126 million sequenced reads, and these were annotated in approximately 80,000 contigs. The search of RNAi-related genes resulted in 47 genes involved in the three major RNAi pathways, with the absence of sid-like homologous. Although ex vivo incubation of dsRNA in E. heros hemolymph showed rapid degradation, there was 35% mortality at 4 days after treatment and a significant reduction in V-ATPase-A gene expression. These results indicated that although sid-like genes are lacking, the dsRNA uptake mechanism was very efficient. Also, 2-fold and 4-fold overexpression of DCR-2 and AGO-2, respectively, after dsRNA supply indicated the activation of the siRNA machinery. Consequently, E. heros has proven to be sensitive to RNAi upon injection of dsRNA into its hemocoel. We believe that this finding together with a publically available transcriptome and the validation of a responsive RNAi machinery provide a starting point for future field applications against one of the most important soybean pests in South America. The Neotropical brown stink bug (BS), Euschistus heros (Hemiptera: Pentatomidae), is one of the most important Pentatomidae pests in South America 1 , especially in soybean (Glycine max) with a reduction in seed quality and yield 2. Stink bugs use their piercing/sucking mouthparts to inject enzymes into the plant tissues to digest plant components and remove pre-digested fluids 3. Although rarely reported before the 70s 2,4 , since then population outbreaks 2,5 and rapid population growth have allowed expansion of the range of E. heros to all the major South American soybean production regions, including Brazil 2 , Paraguay 2 , and Argentina 6 .

Huanglongbing (HLB) is a destructive disease of citrus. The disease is caused by the phloemlimited fastidious proteobacterium Candidatus Liberibacter asiaticus, which is transmitted by the Asian citrus psyllid (Diaphorina citri). The symptoms of HLB have been related by callose accumulation in the phloem sieve plates. The key class of enzymes for callose synthesis is the Callose Synthases. The callose synthase genes (calS) expression is modulated for biotic and abiotic stresses. In this study, nine C. sinensis calS genes (CscalS) were identified and the expression patterns were analyzed in CaLas inoculated and healthy plants. At 120 days after inoculation (dpi), CscalS2 and CscalS7 were significantly up-regulated in the HLB positive plants. At 360 dpi CscalS7 and CscalS12 were significantly up-regulated in the HLB positive plants. Our results confirmed that CaLas infection is accompanied by the increased deposition of callose in the phloem sieve tubes and accumulation of starch in the leaves. It is suggested that the increased deposition of callose in the phloem sieve tubes is a hypersensitivity reaction, inhibiting phloem transport to consequently reducing the bacterial colonization via phloem, what possibly contribute to the starch accumulation in the leaves and the development of HLB symptoms. It is also proposed that CscalS2, CscalS7 and CscalS12 are involved in citrus defense against CaLas, forming a complex in the phloem.

Interactions between insects and plant pathogens have been more enthusiastically studied in the recent decade, especially those relationships which takes the insects as vectors. The spectrum of these interactions ranges from mutualistic to pathogenic. The length of the co-evolutionary process will determine whether a microorganism shares a friend or a foe relationship with its host, and a friendship connection is frequently observed if the coexistence is longer. This review updates knowledge about the morphological, physiological and genetic mechanisms that drive the interaction between 'Candidatus Liberibacter asiaticus' (Las) and its vector, the Asian citrus psyllid, Diaphorina citri. Las is the predominant causal agent of citrus huanglongbing (HLB) disease, the major constrain to citrus production worldwide. This bacterium is transmitted by D. citri, in a propagative-circulative manner during its feeding from plant host. Understanding of the interactions among vector, plant pathogen and host plant are important for the management of this vector-borne disease complex.

Huanglongbing (HLB), caused mainly by ‘Candidatus Liberibacter asiaticus’ (CLas), is the most devastating citrus disease because all commercial species are susceptible. HLB tolerance has been observed in Poncirus trifoliata and their hybrids. A wide-ranging transcriptomic analysis using contrasting genotypes regarding HLB severity was performed to identify the genetic mechanism associated with tolerance to HLB. The genotypes included Citrus sinensis, Citrus sunki, Poncirus trifoliata and three distinct groups of hybrids obtained from crosses between C. sunki and P. trifoliata. According to bacterial titer and symptomatology studies, the hybrids were clustered as susceptible, tolerant and resistant to HLB. In P. trifoliata and resistant hybrids, genes related to specific pathways were differentially expressed, in contrast to C. sinensis, C. sunki and susceptible hybrids, where several pathways were reprogrammed in response to CLas. Notably, a genetic tolerance mechanism was associated with the downregulation of gibberellin (GA) synthesis and the induction of cell wall strengthening. These defense mechanisms were triggered by a class of receptor-related genes and the induction of WRKY transcription factors. These results led us to build a hypothetical model to understand the genetic mechanisms involved in HLB tolerance that can be used as target guidance to develop citrus varieties or rootstocks with potential resistance to HLB.

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The emergence of citrus huanglongbing (HLB) has been a constraint for worldwide citrus growers. HLB disease is associated to species of the biothrophic bacteria Candidatus Liberibacter spp. (CaL). In this study, we assessed the transcriptional status of salicylic acid (SA) genes and associated defenses between two contrasting citrus genotypes during challenge with Ca. Liberibacter asiaticus or Ca. Liberibacter americanus. Citrus sinensis exhibited the most evident alterations in gene expression of evaluated genes, when compared with Poncirus trifoliata. Upstream pathway SA genes showed a slight upward regulation in C. sinensis. Salicylic acid biosynthesis and accumulation might be impaired as we observed a low expression level of SA biosynthesis related genes. Moreover, genes associated to SA metabolism showed a slight induction. These results may account for the absence of significant downstream defense response related to salicylic acid. Leaf anatomical analysis revealed callose accumulation in both HLB infected, C. sinensis and P. trifoliata sieve tube elements (STE), although only C. sinensis exhibited collapsed STE. Our data corroborate other studies and suggest that the SA biosynthesis and metabolism related genes might be involved in the contrasting response to CaL in different citrus genotypes. Additionally, we suggest that collapsed STE might have a prominent implication in symptomatology of highly susceptible plants. Index terms: SAR, plant-pathogen interaction, gene expression, callose deposition. Análises genéticas das respostas de defesas mediadas por ácido salicílicoe histopatologia no patossistema huanglongbing RESUMO A ocorrência do huanglongbing (HLB) dos citros tem sido um sério problema para os citricultores em todo o mundo. O HLB está associado a bactérias biotróficas da espécie Candidatus Liberibacter spp. (CaL). Nesse estudo, nós avaliamos o perfil transcricional dos genes da via do ácido salicílico (SA) e genes associados a defesa em dois genótipos contrastantes após desafio com Ca. Liberibacter asiaticus ou Ca. Liberibacter americanus. Citrus sinensis exibiu maiores alterações na expressão gênica dos genes avaliados, quando comparado com Poncirus trifoliata. Genes upstream da via do Oliveira et al.