Knockdown of calreticulin, laccase, and Snf7 Genes Through RNAi Is Not Effective to Control the Asian Citrus Psyllid (Hemiptera: Livideae).

The Asian citrus psyllid, Diaphorina citri Kuwayama, transmits the bacteria Candidatus Liberibacter associated with huanglongbing (HLB), a devastating disease of the citrus industry. The use of genetically modified plants is an alternative to control this vector. Conversely, technology based on RNA interference (RNAi) for silencing specific genes of a target insect could be attempted. This work evaluated the knockdown effect of the target genes calreticulin (DcCRT), laccase (DcLAC), and Snf7 (DcSnf7) by RNAi through feeding D. citri in Murraya paniculata leaves after the uptake of an aqueous solution with dsRNA homologous to each vector target gene. Confocal microscopy revealed the uptake of the fluorescent-labeled dsRNA by detached leaves and the symplastic movement, allowing the ingestion by the feeding insect. A reduction in the survival rate was observed only 144 h after the beginning of feeding with dsRNA targeting DcSnf7; however, no reduction in transcript accumulation. The knockdown of the DcCRT and DcLAC genes was detected only 12 and 96 h after insect feeding, respectively. Additionally, a reduction in amino acid excretion from insects fed with dsRNA targets to DcCRT and DcLAC was observed 120 h after the beginning of feeding. However, the effects of the dsRNAs tested here appear to be minimal, both at the transcriptional and phenotype levels. For most concentrations and time points, no effects were observed. Therefore, the knockdown of genes DcCRT, DcLAC, and DcSnf7 do not appear to have the potential to control of D. citri through RNAi-mediated gene silencing.

Sugarcane smut: shedding light on the development of the whip-shaped sorus.

Background and Aims: Sugarcane smut is caused by the fungus Sporisorium scitamineum (Ustilaginales/Ustilaginomycotina/Basidiomycota), which is responsible for losses in sugarcane production worldwide. Infected plants show a profound metabolic modification resulting in the development of a whip-shaped structure (sorus) composed of a mixture of plant tissues and fungal hyphae. Within this structure, ustilospores develop and disseminate the disease. Despite the importance of this disease, a detailed histopathological analysis of the plant-pathogen interaction is lacking. Methods: The whip-shaped sorus was investigated using light microscopy, scanning and transmission electron microscopy, histochemical tests and epifluorescence microscopy coupled with deconvolution. Key Results: Sorus growth is mediated by intercalary meristem activity at the base of the sorus, where the fungus causes partial host cell wall degradation and formation of intercellular spaces. Sporogenesis in S. scitamineum is thallic, with ustilospore initials in intercalary or terminal positions, and mostly restricted to the base of the sorus. Ustilospore maturation is centrifugal in relation to the ground parenchyma and occurs throughout the sorus median region. At the apex of the sorus, the fungus produces sterile cells and promotes host cell detachment. Hyphae are present throughout the central axis of the sorus (columella). The plant cell produces callose around the intracellular hyphae as well as inside the papillae at the infection site. Conclusions: The ontogeny of the whip-shaped sorus suggests that the fungus can cause the acropetal growth in the intercalary meristem. The sporogenesis of S. scitamineum was described in detail, demonstrating that the spores are formed exclusively at the base of the whip. Light was also shed on the nature of the sterile cells. The presence of the fungus alters the host cell wall composition, promotes its degradation and causes the release of some peripheral cells of the sorus. Finally, callose was observed around fungal hyphae in infected cells, suggesting that deposition of callose by the host may act as a structural response to fungal infection.

Ultrastructural changes in the epidermis of petals of the sweet orange infected by Colletotrichum acutatum.

Postbloom fruit drop (PFD) is an important disease caused by the fungus Colletotrichum acutatum. PFD is characterised by the formation of necrotic lesions on the petals and stigmas of flowers as well as premature abscission of the fruit in Citrus spp. We compare the ultrastructure of the epidermis of uninoculated Citrus sinensis petals with that of petals inoculated with the fungus to understand the changes that occur upon C. acutatum infection. Healthy petals have a cuticle with parallel striations covering the uniseriate epidermis. This pattern consists of vacuolated parietal cells whose cytoplasm contains mitochondria, plastids with an undeveloped endomembrane system and a slightly dense stroma, a poorly developed rough endoplasmic reticulum, polysomes, few lipid droplets, and a nucleus positioned near the inner periclinal wall. In damaged regions, the cytoplasm of some cells is densely packed with well-developed endoplasmic reticulum, a large number of hyperactive dictyosomes, numerous mitochondria, and many lipid droplets. The plastids have an electron-dense stroma, starch grains, and a large amount of electron-dense lipid droplets, which can be released into vacuoles or the endoplasmic reticulum. Multivesicular bodies and myelin bodies are frequently observed in the vacuole, cytoplasm, and periplasmic space. Vesicles migrate through the cell wall and are involved in the deposition of cuticular material. In the later stages of infection, there is deposition of new cuticle layers in plaques. The outer periclinal walls can be thick. These observations indicate that epidermal cells respond to the pathogen, resulting in cuticular and parietal changes, which may limit further infection.