Nematode Genome Announcement: A Draft Genome of Seed Gall Nematode, Anguina tritici

Anguina tritici is the first plant-parasitic nematode described in literature, dating back to the year 1743. It is responsible for causing earcockle (seed gall) and tundu diseases in wheat and rye. Notably, this nematode has been observed to survive in an anhydrobiotic state for up to 32 years within wheat seed galls. These exceptional characteristics have inspired the sequencing of the A. tritici genome. In this study, we present the initial draft genome of A. tritici, obtained using the Illumina MiSeq platform with coverage of 60-fold. The genome is estimated to have a size of 164 Mb and comprises 39,965 protein-coding genes, exhibiting a GC content of 39.1%. The availability of this genome data will serve as a foundation for future functional biological investigations, particularly for genes whose functions remain unknown to this day

Quantitative detection of pathogen load of Fusarium oxysporum f.sp. ciceris infected wilt resistant and susceptible genotypes of chickpea using intergenic spacer region-based marker

Highlights • Intergenic spacer based markers are robust molecular markers for detection of phytopathogenic fungi. • qPCR can be utilized as a molecular diagnostic tool to quantify pathogen DNA load at pictogram (pg) level. • Differential dynamics of pathogen DNA in chickpea genotypes contrasting for Fusarium wilt resistance was observed.Quantitative detection of pathogen DNA load is a crucial aspect in development of disease management strategies and breeding programs. In recent years, there have been several reports where formae speciales specific intergenic spacer (IGS) sequence based markers have been used for quantification of pathogen DNA in different plant and soil samples, through quantitative real-time PCR (qPCR). In the present study, we have utilized an IGS based marker, ISR 52, to detect and quantify Fusarium oxysporum f.sp. ciceris (Foc) DNA, using both conventional PCR and qPCR, in chickpea genotypes which contrast for resistance to Fusarium wilt. Our study reveals that the Foc DNA load was found to be significantly higher in the early wilting genotypes as compared to the wilt resistant genotypes. Late wilting genotype showed a spike in pathogen DNA load in later stage of plant growth. Phenotypic observation of disease progression in combination with qPCR data validated that the pathogen undergoes incubation period before manifestation of symptoms. The above observations provide evidence about the differential dynamics of pathogen build up inside different hosts during different time periods and probable reason for the earliness, lateness and resistance in wilting like traits in these genotypes

DataSheet_1_Gall-specific promoter, an alternative to the constitutive CaMV35S promoter, drives host-derived RNA interference targeting Mi-msp2 gene to confer effective nematode resistance.docx

One of the major obligate plant parasites causing massive economic crop losses belongs to the class of root-knot nematodes (RKNs). Targeting of major nematode parasitism genes via Host Delivered-RNAi (HD-RNAi) to confer silencing is established as one of the most effective approaches to curb nematode infection. Utilizing nematode-responsive root-specific (NRRS) promoters to design a dsRNA molecule targeting approach to hamper nematode parasitism. Here, a previously validated peroxidase gall specific promoter, pAt2g18140, from Arabidopsis was employed to express the dsRNA construct of the nematode effector gene Mi-msp2 from Meloidogyne incognita. Arabidopsis RNAi lines of CaMV35S::Mi-msp2-RNAi and pAt2g18140::Mi-msp2-RNAi were compared with control plants to assess the decrease in plant nematode infection. When subjected to infection, the maximum reductions in the numbers of galls, females and egg masses in the CaMV35S::Mi-msp2-RNAi lines were 61%, 66% and 95%, respectively, whereas for the pAt2g18140::Mi-msp2-RNAi lines, they were 63%, 68% and 100%, respectively. The reduction in transcript level ranged from 79%-82% for CaMV35S::Mi-msp2-RNAi and 72%-79% for the pAt2g18140::Mi-msp2-RNAi lines. Additionally, a reduction in female size and a subsequent reduction in next-generation fecundity demonstrate the efficacy and potential of the gall specific promoter pAt2g18140 for utilization in the development of HD-RNAi constructs against RKN, as an excellent alternative to the CaMV35S promoter.</p

Identification and Characterization of Wilt and Salt Stress-Responsive MicroRNAs in Chickpea through High-Throughput Sequencing

<div><p>Chickpea (<i>Cicer arietinum</i>) is the second most widely grown legume worldwide and is the most important pulse crop in the Indian subcontinent. Chickpea productivity is adversely affected by a large number of biotic and abiotic stresses. MicroRNAs (miRNAs) have been implicated in the regulation of plant responses to several biotic and abiotic stresses. This study is the first attempt to identify chickpea miRNAs that are associated with biotic and abiotic stresses. The wilt infection that is caused by the fungus <i>Fusarium oxysporum</i> f.sp. <i>ciceris</i> is one of the major diseases severely affecting chickpea yields. Of late, increasing soil salinization has become a major problem in realizing these potential yields. Three chickpea libraries using fungal-infected, salt-treated and untreated seedlings were constructed and sequenced using next-generation sequencing technology. A total of 12,135,571 unique reads were obtained. In addition to 122 conserved miRNAs belonging to 25 different families, 59 novel miRNAs along with their star sequences were identified. Four legume-specific miRNAs, including miR5213, miR5232, miR2111 and miR2118, were found in all of the libraries. Poly(A)-based qRT-PCR (Quantitative real-time PCR) was used to validate eleven conserved and five novel miRNAs. miR530 was highly up regulated in response to fungal infection, which targets genes encoding zinc knuckle- and microtubule-associated proteins. Many miRNAs responded in a similar fashion under both biotic and abiotic stresses, indicating the existence of cross talk between the pathways that are involved in regulating these stresses. The potential target genes for the conserved and novel miRNAs were predicted based on sequence homologies. miR166 targets a HD-ZIPIII transcription factor and was validated by 5′ RLM-RACE. This study has identified several conserved and novel miRNAs in the chickpea that are associated with gene regulation following exposure to wilt and salt stress.</p></div

Length distribution of small RNA population.

<p>Size distributions of the miRNAs in the three chickpea libraries. In the wilt stress library, 20 nt miRNAs are more frequent than 24 nt miRNAs. However, in the other two libraries 24 nt miRNAs are more frequent.</p

Mapping of target mRNA cleavage site of miR166 by modified 5′ RACE.

<p>The target of miR166 (TC04758) encodes a transcription factor belonging to class III of the HD-ZIP family protein. The arrow indicates the cleavage site, and the numbers above the arrow denote the frequencies of the sequenced clones.</p

Expression analyses of selected miRNAs under wilt and salt stresses as evaluated by qRT-PCR.

<p>The relative expression levels are shown as fold changes with the standard errors (SE) of three biological replicates. (A) Expression profiling of conserved miRNAs under control, wilt and salt stress conditions. (B) Expression profiling of novel miRNAs under control, wilt and salt stress conditions.</p

Distribution of the sequenced reads in the control, wilt- and salt-stressed chickpea libraries.

<p>Distribution of the sequenced reads in the control, wilt- and salt-stressed chickpea libraries.</p

Multiple sequence alignments of legume-specific miRNAs. (A) Mature miRNAs, (B) precursor miRNAs.

<p>Four legume-specific miRNAs, including a) MIR5213, b) MIR5232, c) MIR2111and d) MIR2118, were used for the multiple sequence alignments by ClustalW2 in the different plants. car- <i>Cicer arietinum</i>, mtr- <i>Medicago truncatula</i>, gma- <i>Glycine max</i>, ath- <i>Arabidopsis thaliana</i>, osa- <i>Oryza sativa</i>, zma- <i>Zea mays</i>, sbi- <i>Sorghum bicolor</i>, sly- <i>Solanum lycopersicum</i>, hbr- <i>Hevea brasiliensis</i>, pvu<i>- Phaseolus vulgaris</i>, vun- <i>Vigna unguiculata</i>, ptc- <i>Populus trichocarpa</i> and mdm- <i>Malus domestica.</i></p

Gene ontology categories of predicted target transcripts for chickpea miRNAs.

<p>The miRNA-target genes were categorized according to the molecular function, biological process and cellular component sub-ontologies.</p