Omnipresence of Partitiviruses in Rice Aggregate Sheath Spot Symptom-Associated Fungal Isolates from Paddies in Thailand

Partitiviruses are one of the most prevalent double-stranded RNA viruses that have been identified mostly in filamentous fungi and plants. Partitiviruses generally infect host fungi asymptomatically but infrequently exert significant effect(s) on morphology and virulence, thus being considered a potential source of biological control agents against pathogenic fungi. In this study, we performed a screening for mycoviruses of a collection of Thai isolates of rice fungal pathogen Rhizoctonia oryzae-sativae, a causal agent of rice aggregated sheath spot disease. As a result, 36% of tested isolates carried potentially viral double-stranded RNAs with sizes ranging from 2 to 3 kbp. By conventional cDNA library construction and RNA-seq, we determined six new alphapartitiviruses that infected three isolates: tentatively named Rhizoctonia oryzae-sativae partitivirus 1 to 6 (RosPV1-6). Furthermore, RT-PCR detection of each virus revealed their omnipresent nature in different R. oryzae-sativae isolates. Although virus-curing of basidiomycetous fungi is generally difficult, our repeated attempts successfully obtained virus-free (for RosPV1, RosPV2, and uncharacterized partitiviruses), isogenic strain of R. oryzae-sativae TSS190442. The virus-cured strain showed slightly faster colony growth on the synthetic media and severe symptom development on the rice sheath compared to its virus-infected counterpart. Overall, this study shed light on the distribution of partitiviruses in R. oryzae-sativae in a paddy environment and exemplified a virus-curing protocol that may be applicable for other basidiomycetous fungi.

Strandedness During cDNA Synthesis, the Stranded Parameter in htseq-count, and Analysis of RNA-Seq Data

RNA sequencing (RNA-Seq) is a complicated protocol, both in the laboratory in generation of data and at the computer in analysis of results. Several decisions during RNA-Seq library construction have important implications for analysis, most notably strandedness during complementary DNA (cDNA) library construction. Here we clarify bioinformatic decisions related to strandedness in both alignment of DNA sequencing reads to reference genomes and subsequent determination of transcript abundance.

cDNA library construction of two human Demodexspecies

The research of

Konstruksi pustaka cDNA dari daun klon karet AVROS 2037 yang diinfeksi patogen Corynespora cassiicola Construction of a cDNA library from leaf of AVROS 2037 rubber clone infected by Corynespora cassiicola pathogen

SummaryConstruction of cDNA library derived fromtranscripts made under certain condition is animportant first step to understand diseaseresistant mechanisms. To identify rubber genesor transcripts involved in defense responsetoward Corynespora cassiicola, cDNA librarywas constructed using rubber clone AVROS2037, one of resistant clone to this pathogen.cDNA library was constructed based on thestrategy of leaves infection using conidia, withthe assumption that transcript expression relatedto defense response would be induced bypathogen infection. RNA was isolated from leavesthree days after inoculation with conidia ofC. cassiicola. Steps involved in the cDNA libraryconstruction were RNA isolation, mRNApurification, cDNA synthesis, vector modifcation,cDNA insert ligation, plasmid transformation andclone verifications. Each gram of leaf producedapproximately 300  g RNA, and 0.25% of themwas mRNA. The mRNA was used to synthesizedcDNA. Ligation of cDNA and modified vectorwas facilitated by restriction enzyme SfiI. Theconstructs were transformed into the E. coliDH5 competent cells. A total of 8000 colonieswere produced. Random examination of 270colonies showed that approximately 93% of thesecolonies carried plasmid vector with DNA insertsize of 200 – 2000 bp, with average size of 500 –800 bp. cDNA library construction of rubberleaves from AVROS 2037 clone as well as somenecessary modification steps are presented in thispaper.RingkasanKonstruksi pustaka cDNA yang me-ngandung transkrip yang diekspresikan dalamkondisi tertentu merupakan tahap awal yangsangat penting dalam berbagai studi biologi.Untuk mengidentifikasi gen karet atau transkripyang berperan dalam respons pertahanan tanamankaret terhadap Corynespora cassiicola, pustakacDNA dibuat dengan menggunakan daun klonAVROS 2037 yang merupakan salah satu klonresisten terhadap patogen tersebut. PustakacDNA dibuat berdasarkan strategi menginfeksidaun dengan konidia C. cassiicola denganpertimbangan bahwa ekspresi transkrip yangberperan dalam respons pertahanan akandiinduksi oleh adanya infeksi patogen. Dengandemikian pustaka cDNA yang dibuat diharapkanmengandung gen atau bagian gen yang ber-hubungan dengan respons pertahanan. RNAdiisolasi dari daun setelah daun diinokulasiselama tiga hari dengan konidia C. cassiicola.Beberapa tahapan telah dilakukan, dimulaidengan isolasi RNA, pemurnian mRNA, sintesiscDNA, modifikasi vektor kloning, ligasi fragmencDNA utas ganda dengan vektor kloning sertatransformasi hasil ligasi ke bakteri Escherichiacoli DH5 kompeten. Dari setiap gram jaringandaun berhasil diisolasi RNA sekitar 300 g, dandari jumlah tersebut sekitar 0,25% mRNA dapatdiisolasi. mRNA yang diisolasi digunakan untuksintesis cDNA. cDNA dipotong dengan enzimrestriksi SfiI dan diligasi ke vektor plasmid yangdimodifikasi dengan menyisipkan situs enzimSfiI. cDNA-vektor rekombinan ditransformasi kedalam sel bakteri E. coli DH5 kompeten meng-gunakan metode standar. Transformasi konstrukini menghasilkan 8.000 koloni. Pengujian PCRterhadap 270 koloni yang dipilih secara acakmengindikasikan bahwa sekitar 93% kolonitersebut membawa cDNA sisipan dengan ukuranfragmen cDNA yang menyisip berkisar antara200 sampai 2000 bp. cDNA sisipan terbanyakterdapat pada ukuran antara 500 – 800 bp. Dalamtulisan ini dibahas tahap demi tahap proses yangdilakukan untuk membuat pustaka cDNA asaldaun karet klon AVROS 2037 serta beberapamodifikasi yang diperlukan.