The complete mitochondrial genome of Leucoptera coffeella (Lepidoptera: Lyonetiidae) and phylogenetic relationships within the Yponomeutoidea superfamily.

The coffee leaf miner (Leucoptera coffeella) is one of the major pests of coffee crops in the neotropical regions, and causes major economic losses. Few molecular data are available to identify this pest and advances in the knowledge of the genome of L. coffeella will contribute to improving pest identification and also clarify taxonomy of this microlepidoptera. L. coffeella DNA was extracted and sequenced using PacBio HiFi technology. Here we report the complete L. coffeella circular mitochondrial genome (16,407 bp) assembled using Aladin software. We found a total of 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs) and an A + T rich-region and a D-loop. The L. coffeella mitochondrial gene organization is highly conserved with similarities to lepidopteran mitochondrial gene rearrangements (trnM-trnI-trnQ). We concatenated the 13 PCG to construct a phylogenetic tree and inferred the relationship between L. coffeella and other lepidopteran species. L. coffeella is found in the Lyonetiidae clade together with L. malifoliella and Lyonetia clerkella, both leaf miners. Interestingly, this clade is assigned in the Yponomeutoidea superfamily together with Gracillariidae, and both superfamilies displayed species with leaf-mining feeding habits.

In planta RNAi approach targeting three M. incognita effector genes disturbed the process of infection and reduced plant susceptibility.

Meloidogyne incognita is the most economically important species of the root-knot nematode complex causing damage to several crops worldwide. During parasitism in host plants, M. incognita secretes several effector proteins to suppress the plant immune system, manipulate the plant cell cycle, and promote parasitism. Several effector proteins have been identified, but their relationship with plant parasitism by M. incognita has not been fully confirmed. Herein, the Minc01696, Minc00344, and Minc00801 putative effector genes were evaluated to assess their importance during soybean and Nicotiana tabacum parasitism by M. incognita. For this study, we used in planta RNAi technology to overexpress dsRNA molecules capable of producing siRNAs that target and downregulate these nematode effector genes. Soybean composite roots and N. tabacum lines were successfully generated, and susceptibility level to M. incognita was evaluated. Consistently, both transgenic soybean roots and transgenic N. tabacum lines carrying the RNAi strategy showed reduced susceptibility to M. incognita. The number of galls per plant and the number of egg masses per plant were reduced by up to 85% in transgenic soybean roots, supported by the downregulation of effector genes in M. incognita during parasitism. Similarly, the number of galls per plant, the number of egg masses per plant, and the nematode reproduction factor were reduced by up to 83% in transgenic N. tabacum lines, which was also supported by the downregulation of the Minc00801 effector gene during parasitism. Therefore, our data indicate that all three effector genes can be a target in the development of new biotechnological tools based on the RNAi strategy in economically important crops for M. incognita control.

The Identification of Small RNAs Differentially Expressed in Apple Buds Reveals a Potential Role of the Mir159-MYB Regulatory Module during Dormancy.

Winter dormancy is an adaptative mechanism that temperate and boreal trees have developed to protect their meristems against low temperatures. In apple trees (Malus domestica), cold temperatures induce bud dormancy at the end of summer/beginning of the fall. Apple buds stay dormant during winter until they are exposed to a period of cold, after which they can resume growth (budbreak) and initiate flowering in response to warmer temperatures in spring. It is well-known that small RNAs modulate temperature responses in many plant species, but however, how small RNAs are involved in genetic networks of temperature-mediated dormancy control in fruit tree species remains unclear. Here, we have made use of a recently developed ARGONAUTE (AGO)-purification technique to isolate small RNAs from apple buds. A small RNA-seq experiment resulted in the identification of 17 micro RNAs (miRNAs) that change their pattern of expression in apple buds during dormancy. Furthermore, the functional analysis of their predicted target genes suggests a main role of the 17 miRNAs in phenylpropanoid biosynthesis, gene regulation, plant development and growth, and response to stimulus. Finally, we studied the conservation of the Arabidopsis thaliana regulatory miR159-MYB module in apple in the context of the plant hormone abscisic acid homeostasis.

Comparative Genomics Reveals Novel Target Genes towards Specific Control of Plant-Parasitic Nematodes.

Plant-parasitic nematodes cause extensive annual yield losses to worldwide agricultural production. Most cultivated plants have no known resistance against nematodes and the few bearing a resistance gene can be overcome by certain species. Chemical methods that have been deployed to control nematodes have largely been banned from use due to their poor specificity and high toxicity. Hence, there is an urgent need for the development of cleaner and more specific control methods. Recent advances in nematode genomics, including in phytoparasitic species, provide an unprecedented opportunity to identify genes and functions specific to these pests. Using phylogenomics, we compared 61 nematode genomes, including 16 for plant-parasitic species and identified more than 24,000 protein families specific to these parasites. In the genome of Meloidogyne incognita, one of the most devastating plant parasites, we found ca. 10,000 proteins with orthologs restricted only to phytoparasitic species and no further homology in protein databases. Among these phytoparasite-specific proteins, ca. 1000 shared the same properties as known secreted effectors involved in essential parasitic functions. Of these, 68 were novel and showed strong expression during the endophytic phase of the nematode life cycle, based on both RNA-seq and RT-qPCR analyses. Besides effector candidates, transcription-related and neuro-perception functions were enriched in phytoparasite-specific proteins, revealing interesting targets for nematode control methods. This phylogenomics analysis constitutes a unique resource for the further understanding of the genetic basis of nematode adaptation to phytoparasitism and for the development of more efficient control methods.

Mitogenome sequence accuracy using different elucidation methods.

Mitogenome sequences are highly desired because they are used in several biological disciplines. Their elucidation has been facilitated through the development of massive parallel sequencing, accelerating their deposition in public databases. However, sequencing, assembly and annotation methods might induce variability in their quality, raising concerns about the accuracy of the sequences that have been deposited in public databases. In this work we show that different sequencing methods (number of species pooled in a library, insert size and platform) and assembly and annotation methods generated variable completeness and similarity of the resulting mitogenome sequences, using three species of predaceous ladybird beetles as models. The identity of the sequences varied considerably depending on the method used and ranged from 38.19 to 90.1% for Cycloneda sanguinea, 72.85 to 91.06% for Harmonia axyridis and 41.15 to 93.60% for Hippodamia convergens. Dissimilarities were frequently found in the non-coding A+T rich region, but were also common in coding regions, and were not associated with low coverage. Mitogenome completeness and sequence identity were affected by the sequencing and assembly/annotation methods, and high within-species variation was also found for other mitogenome depositions in GenBank. This indicates a need for methods to confirm sequence accuracy, and guidelines for verifying mitogenomes should be discussed and developed by the scientific community.