Detection of Avirulence Gene AvrPi9 in Magnaporthe oryzae, a Rice Blast Fungus, Using a Combination of RPA and CRISPR-Cas12a Techniques

Rice blast disease is one of the most devastating diseases of rice production worldwide, which causes by an ascomycete fungus, Magnaporthe oryzae. The virulence of the rice blast fungus is determined by avirulence genes (Avr genes). Therefore, the identification of Avr genes is important for rice resistance variety improvement. Avr genes are currently identified using the pathogenicity assay with rice near-isogenic lines (NILs) or PCR amplification and gene sequencing, both of which are time-consuming and labor-intensive methods. This study aims to develop a simple method for Avr gene identification using AvrPi9 as a model. A recombinase polymerase amplification (RPA) technique was carried out to amplify AvrPi9 by incubating rice blast fungus genomic DNA with gene-specific primers at 37°C for 20 min. Cas12a-based AvrPi9 detection was performed by incubating at 37°C for 5 min. The fluorescence signal was visualized by the naked eye under an LED transilluminator. The study found that AvrPi9 can be amplified and detected using RPA and a Cas12a-based method. AvrPi9_crRNA2 has a higher efficiency than AvrPi9_crRNA1. The sensitivity of the method was 3.8 ng of DNA target for AvrPi9_crRNA1 and 1.9 ng of DNA target for AvrPi9_crRNA2. This RPA and Cas12a combination technique is a newer method for Avr gene detection in plants and has several advantages over traditional methods. It is considered easier to use and more efficient in terms of time and labor, making it a potentially useful tool for plant breeders and pathologists

Leaf Spot Characteristics of Phomopsis Durionis on Durian (Durio Zibethinus Murray) and Latent Infection of the Pathogen

A survey of leaf spot disease on durian caused by Phomopsis durionis was conducted in durian growing areas in eastern Thailand, Chanthaburi and Trat provinces. It was found that lesions with yellow halos on both mature and young leaves are variable in sizes (1–10 mm in diameter). In this study, nine morphologically distinct isolates of Phomopsis were obtained from durian leaf spots. Some of them produced small number of pycnidia on potato dextrose agar after incubation for 30 days. Artificial inoculation on wounded leaves of durian seedlings, resulted in the production of browning areas with yellow halos and pycnidium formation at 13 days and 20 days after inoculation, respectively. Furthermore, red-brown spots with yellow halos on leaf tissues were observed at 32 days after inoculation. High density of Phomopsis was observed in durian symptomless leaves and flowers indicated the latent infection of the pathogen in the fields. Interestingly, crude extract of durian leaf with preformed substances demonstrated inhibition of spore germination and germ tube growth of the pathogen, Phomopsis sp., on water agar. In addition, bioassay on TLC plate displayed inhibition zone of growth of the fungus, Cladosporium oxysporum at retention factor (Rf) of 0.29–0.88. This indicates that preformed substances in leaf tissues might act as compounds affecting latent period of pathogen

Genetic Diversity and Aggressiveness of Bipolaris oryzae in North-Central Thailand

One hundred and ten isolates of Bipolaris oryzae, the causal agent of rice brown spot disease were collected from paddy fields in four provinces of north-central Thailand, including Ang Thong, Chai Nat, Lop Buri, and Sing Buri. DNA polymorphism of some Bipolaris oryzae isolates was determined by VNTR, ISSR and RAPD markers. Only VNTR-MR primer showed different fingerprint patterns among these isolates, therefore this primer was selected to study genetic diversity of the Bipolaris oryzae population. In total, there were three haplotypes corresponding to the results from cluster analysis; each of the three clusters shared identical haplotype. The majority of the isolates were separated into group A (88.18%), indicating predominant asexual reproduction of clonal population. However, there was no relationship between haplotype and either collection provinces or aggressiveness on rice. Among four rice varieties tested, including Khao Dawk Mali 105 (KDML 105), RD31, Pathum Thani 1, and Jao Hom Nin (JHN), JHN was the most resistant variety, while KDML 105 was the most susceptible to brown spot disease

A Versatile Zero Background T-Vector System for Gene Cloning and Functional Genomics1[C][W][OA]

With the recent availability of complete genomic sequences of many organisms, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. Although site-specific recombination-based cloning systems, such as Gateway cloning technology, are extremely useful for efficient transfer of DNA fragments into multiple destination vectors, the two-step cloning process is time consuming and expensive. Here, we report a zero background TA cloning system that provides simple and high-efficiency direct cloning of PCR-amplified DNA fragments with almost no self-ligation. The improved T-vector system takes advantage of the restriction enzyme XcmI to generate a T-overhang after digestion and the negative selection marker gene ccdB to eliminate the self-ligation background after transformation. We demonstrate the feasibility and flexibility of the technology by developing a set of transient and stable transformation vectors for constitutive gene expression, gene silencing, protein tagging, protein subcellular localization detection, and promoter fragment activity analysis in plants. Because the system can be easily adapted for developing specialized expression vectors for other organisms, zero background TA provides a general, cost-efficient, and high-throughput platform that complements the Gateway cloning system for gene cloning and functional genomics

Genetic Variation of Coleosporium plumeriae from Different Provinces in Thailand

Plumeria rust samples were collected from five provinces in Thailand, including Bangkok, Nakhon Pathom, Rayong, Chonburi and Yala. All five isolates produced the uredial stage but only the isolates from Bangkok and Yala also underwent the telial and basidial stages. The morphological characteristics of all three stages present in the life cycle of the isolates were studied under stereo, compound and electron microscopes. Ribosomal DNA (rDNA) sequences at 28S and ITS (internal transcribed spacer) regions were analyzed with those in the GenBank database by Nucleotide BLAST and phylogenetic analyses. Coleosporium plumeriae was identified as the causal agent of plumeria rust by structure morphology and rDNA sequences that revealed genetic variation of the fungus as well. In general, there were significant differences in the morphological characteristics of uredospores, teliospores and basidia among the isolates. However, the variation of spore morphology was not related to the sampling locations. According to the phylogenetic analysis of 28S rDNA sequences, the UPGMA tree grouped all C. plumeriae from Thailand and foreign countries in the same clade as they shared identical sequences. On the other hand, the UPGMA tree inferred from ITS rDNA sequence data detected genetic variation of the isolate from Chonburi and separated it into the distinct tree branch. In this study, structure morphology and ITS rDNA were suitable genetic markers for both interspecific and intraspecific taxonomy of C. plumeriae

The E3 Ligase APIP10 Connects the Effector AvrPiz-t to the NLR Receptor Piz-t in Rice

<div><p>Although nucleotide-binding domain, leucine-rich repeat (NLR) proteins are the major immune receptors in plants, the mechanism that controls their activation and immune signaling remains elusive. Here, we report that the avirulence effector AvrPiz-t from <i>Magnaporthe oryzae</i> targets the rice E3 ligase APIP10 for degradation, but that APIP10, in return, ubiquitinates AvrPiz-t and thereby causes its degradation. Silencing of <i>APIP10</i> in the non-<i>Piz-t</i> background compromises the basal defense against <i>M</i>. <i>oryzae</i>. Conversely, silencing of <i>APIP10</i> in the <i>Piz-t</i> background causes cell death, significant accumulation of Piz-t, and enhanced resistance to <i>M</i>. <i>oryzae</i>, suggesting that APIP10 is a negative regulator of Piz-t. We show that APIP10 promotes degradation of Piz-t via the 26S proteasome system. Furthermore, we demonstrate that AvrPiz-t stabilizes Piz-t during <i>M</i>. <i>oryzae</i> infection. Together, our results show that APIP10 is a novel E3 ligase that functionally connects the fungal effector AvrPiz-t to its NLR receptor Piz-t in rice.</p></div

AvrPiz-t and APIP10 degrade each other in <i>N</i>. <i>benthamiana</i>.

<p>(<b>A</b>) Degradation of GFP:AvrPiz-t:HA with co-expression of Myc:APIP10 in <i>N</i>. <i>benthamiana</i>. Myc:APIP10 or Myc:APIP10 dRING was co-expressed with GFP:AvrPiz-t:HA by agro-infiltration. Tissues were harvested 2 days after infiltration. MG132 (50 μM) was infiltrated with DMSO as a control at 18 h before sampling. Tap tag protein was expressed as an internal control and was detected by immunoblot with the peroxidase anti-peroxidase (PAP). The transcriptional level of each gene was determined by semi-quantitative (sq)-PCR. dRING denotes APIP10 dRING. The experiments were performed at least three times with similar results. (<b>B</b>) Degradation of APIP10 when it is co-expressed with the AvrPiz-t protein in <i>N</i>. <i>benthamiana</i>. Tissues were harvested 3 days after infiltration. MG132 (50 μM) was infiltrated with DMSO as a control at 18 h before sampling. The experiments were performed at least three times with similar results.</p