Degradome comparison between wild and cultivated rice identifies differential targeting by miRNAs

Background Small non-coding (s)RNAs are involved in the negative regulation of gene expression, playing critical roles in genome integrity, development and metabolic pathways. Targeting of RNAs by ribonucleoprotein complexes of sRNAs bound to Argonaute (AGO) proteins results in cleaved RNAs having precise and predictable 5` ends. While tools to study sliced bits of RNAs to confirm the efficiency of sRNA-mediated regulation are available, they are sub-optimal. In this study, we provide an improvised version of a tool with better efficiency to accurately validate sRNA targets. Results Here, we improvised the CleaveLand tool to identify additional micro (mi)RNA targets that belong to the same family and also other targets within a specified free energy cut-off. These additional targets were otherwise excluded during the default run. We employed these tools to understand the sRNA targeting efficiency in wild and cultivated rice, sequenced degradome from two rice lines, O. nivara and O. sativa indica Pusa Basmati-1 and analyzed variations in sRNA targeting. Our results indicate the existence of multiple miRNA-mediated targeting differences between domesticated and wild species. For example, Os5NG4 was targeted only in wild rice that might be responsible for the poor secondary wall formation when compared to cultivated rice. We also identified differential mRNA targets of secondary sRNAs that were generated after miRNA-mediated cleavage of primary targets. Conclusions We identified many differentially targeted mRNAs between wild and domesticated rice lines. In addition to providing a step-wise guide to generate and analyze degradome datasets, we showed how domestication altered sRNA-mediated cascade silencing during the evolution of indica rice.

Exploring the Genetic Diversity among Weedy Rice Accessions Differing in Herbicide Tolerance and Allelopathic Potential

Increasing agricultural productivity is indispensable to meet future food demand. Crop improvement programs rely heavily on genetic diversity. The success of weeds in the ecosystem can be attributed to genetic diversity and plasticity. Weedy rice, a major weed of rice, has diverse morphology and phenology, implying wide genetic diversity. Study was conducted to genotype weedy rice accessions (n = 54) previously phenotyped for herbicide tolerance and allelopathic potential using 30 SSR markers. Cultivated rice (CL163, REX) and allelopathic rice (RONDO, PI312777, PI338047) were also included in the study. Nei’s genetic diversity among weedy rice (0.45) was found to be higher than cultivated rice (0.24) but less than allelopathic rice (0.56). The genetic relationship and population structure based on herbicide tolerance and allelopathic potential were evaluated. Herbicide-tolerant and susceptible accessions formed distinct clusters in the dendrogram, indicating their genetic variation, whereas no distinction was observed between allelopathic and non-allelopathic weedy rice accessions. Weedy rice accession B2, which was previously reported to have high allelopathy and herbicide tolerance, was genetically distinct from other weedy rice. Results from the study will help leverage weedy rice for rice improvement programs as both rice and weedy rice are closely related, thus having a low breeding barrier.

Flooding depths and burial effects on seedling emergence of five California weedy rice (Oryza sativa spontanea) accessions

Weedy rice (Oryza sativa f. spontanea Roshev.) has recently become a significant botanical pest in California rice (Oryza sativa L.) production systems. The conspecificity of this pest with cultivated rice, Oryza sativa (L.), negates the use of selective herbicides, rendering the development of non-chemical methods a necessary component of creating management strategies for this weed. Experiments were conducted to determine the emergence and early growth responses of O. sativa spontanea to flooding soil and burial conditions. Treatment combinations of four flooding depths (0, 5, 10, and 15 cm) and four burial depths (1.3, 2.5, 5, and 10 cm) were applied to test the emergence of five O. sativa spontanea accessions as well as ‘M-206’, a commonly used rice cultivar in California, for comparison. Results revealed that burial depth had a significant effect on seedling emergence. There was a 43-91% decrease in emergence between seedlings buried at 1.3 and 2.5 cm depending on the flooding depth and accession, and an absence of emergence from seedlings buried at or below 5 cm. Flooding depth did not affect emergence, but there was a significant interaction between burial and flooding treatments. There was no significant difference between total O. sativa spontanea emergence from the soil and water surfaces regardless of burial or flooding depths, implying that once the various accessions have emerged from the soil they will also emerge from the floodwater. Most accessions had similar total emergence compared to M-206 cultivated rice, but produced more dry weight than M-206 when planted at 1.3 cm in the soil. The results of this experiment can be used to inform stakeholders of the flooding conditions necessary as well as soil burial depths that will promote or inhibit the emergence of California O. sativa spontanea accessions from the weed seedbank.

Photosynthetic traits of Australian wild rice (Oryza australiensis) confer tolerance to extreme daytime temperatures

Key message A wild relative of rice from the Australian savannah was compared with cultivated rice, revealing thermotolerance in growth and photosynthetic processes and a more robust carbon economy in extreme heat. Abstract Above ~ 32 °C, impaired photosynthesis compromises the productivity of rice. We compared leaf tissues from heat-tolerant wild rice (Oryza australiensis) with temperate-adapted O. sativa after sustained exposure to heat, as well as diurnal heat shock. Leaf elongation and shoot biomass in O. australiensis were unimpaired at 45 °C, and soluble sugar concentrations trebled during 10 h of a 45 °C shock treatment. By contrast, 45 °C slowed growth strongly in O. sativa. Chloroplastic CO2 concentrations eliminated CO2 supply to chloroplasts as the basis of differential heat tolerance. This directed our attention to carboxylation and the abundance of the heat-sensitive chaperone Rubisco activase (Rca) in each species. Surprisingly, O. australiensis leaves at 45 °C had 50% less Rca per unit Rubisco, even though CO2 assimilation was faster than at 30 °C. By contrast, Rca per unit Rubisco doubled in O. sativa at 45 °C while CO2 assimilation was slower, reflecting its inferior Rca thermostability. Plants grown at 45 °C were simultaneously exposed to 700 ppm CO2 to enhance the CO2 supply to Rubisco. Growth at 45 °C responded to CO2 enrichment in O. australiensis but not O. sativa, reflecting more robust carboxylation capacity and thermal tolerance in the wild rice relative.

A More Complete Story of the Genetic Bases of Resistance to the Rice Hoja Blanca Virus

Rice hoja blanca is one of the most serious diseases in rice growing areas in tropical Americas. Its causal agent is the Rice hoja blanca virus (RHBV), transmitted by the planthopper Tagosodes orizicolus Müir. Genetic resistance is the most effective and environment-friendly way of controlling the disease. So far, only one major quantitative trait locus (QTL) of Oryza sativa ssp. japonica origin, qHBV4.1, that alters incidence of the virus symptoms in two Colombian cultivars has been reported. This resistance has already started to be broken, stressing the urgent need for diversifying the resistance sources. In the present study we performed a search for new QTLs of O. sativa indica origin associated with RHBV resistance. We used four F2:3 segregating populations derived from indica resistant varieties crossed with a highly susceptible japonica pivot parent. Beside the standard method for measuring disease incidence, we developed a new method based on computer-assisted image processing to determine the affected leaf area (ALA) as a measure of symptoms severity. Based on the disease severity and incidence scores in the F3 families under greenhouse conditions, and SNP genotyping of the F2 individuals, we identified four new indica QTLs for RHBV resistance on rice chromosomes 4, 6 and 11, namely qHBV4.2WAS208, qHBV6.1PTB25, qHBV11.1 and qHBV11.2. We also confirmed the wide-range action of qHBV4.1. Among the five QTLs, qHBV4.1 and qHBV11.1 had the largest effects on incidence and severity, respectively. These results provide a more complete understanding of the genetic bases of RHBV resistance in the cultivated rice gene pool, and can be used to develop marker-aided breeding strategies to improve RHBV resistance. The power of joint- and meta- analyses allowed precise mapping and candidate genes identification, providing the basis for positional cloning of the two major QTLs qHBV4.1 and qHBV11.1.

An Allelic Variant of the Broad-Spectrum Blast Resistance Gene Ptr in Weedy Rice is Associated with Resistance to the Most Virulent Blast Race IB-33

Rice resistance (R) genes have been effectively deployed to prevent blast disease caused by the pathogen Magnaporthe oryzae, one of the most serious threats for stable rice production worldwide. Weedy rice competing with cultivated rice may carry novel or lost R genes. The QTL qBR12.3b was previously mapped between two single nucleotide polymorphism (SNP) markers 10,633,942 bp and 10,820,033 bp in a black hull awned (BHA) weed strain using a weed-crop mapping population under greenhouse conditions. In the present study, we found a portion of the known resistance gene Ptr encoding a protein with 4 armadillo repeats and confers a broad spectrum of blast resistance. We then analyzed the sequences of the Ptr gene from weedy rice, PtrBHA, identified a unique amino acid glutamine (Gln) at protein position 874. Minor changes of protein conformation of the PtrBHA gene were predicted through structural analysis of PtrBHA suggesting the product of PtrBHA is involved in disease resistance. A gene-specific codominant marker HJ17-13 from PtrBHA was then developed to distinguish alleles in weed and crop. The existence of the PtrBHA gene in 207 individuals of the same mapping population where qBR12.3b was mapped using this gene-specific marker. Disease reactions of 207 individuals and their parents to IB-33 were evaluated. The resistant individuals had the PtrBHA whereas the susceptible individuals did not suggest HJ17-13 is reliable to predict qBR12.3b. Taken together, this newly developed marker and weedy rice genotypes carrying qBR12.3b are useful for blast improvement using marker assisted selection.

Control of Thousand-Grain Weight by OsMADS56 in Rice

Grain weight and size are important traits determining grain yield and influencing grain quality in rice. In a previous study, a quantitative trait locus controlling thousand-grain weight (TGW) in rice, qTGW10-20.8, was mapped in a 70.7 kb region on chromosome 10. Validation of the candidate gene for qTGW10-20.8, OsMADS56 encoding a MADS-box transcription factor, was performed in this study. In a near-isogenic line (NIL) population segregated only at the OsMADS56 locus, NILs carrying the OsMADS56 allele of IRBB52 were 1.9% and 2.9% lower in TGW than NILs carrying the OsMADS56 allele of Teqing in 2018 and 2020, respectively. Using OsMADS56 knock-out mutants and overexpression transgenic plants, OsMADS56 was validated as the causal gene for qTGW10-20.8. Compared with the recipients, the TGW of the mutants was reduced by 6.0–15.0%. In these populations, decreased grain weight and size were associated with a reduction in the expression of OsMADS56. In transgenic populations of OsMADS56 driven by a strong constitutive promoter, grain weight and size of the positive plants were significantly higher than those of the negative plants. Haplotype analysis showed that the Teqing-type allele of OsMADS56 is the major type presented in cultivated rice and used in variety improvement. Cloning of OsMADS56 provides a new gene resource to improve grain weight and size through molecular design breeding.

Vacuolar Protein-Sorting Receptor MoVps13 Regulates Conidiation and Pathogenicity in Rice Blast Fungus Magnaporthe oryzae

Magnaporthe oryzae (synonym Pyricularia oryzae) is a filamentous fungal pathogen that causes major yield losses in cultivated rice worldwide. However, the mechanisms of infection of M. oryzae are not well characterized. The VPS13 proteins play vital roles in various biological processes in many eukaryotic organisms, including in the organization of actin cytoskeleton, vesicle trafficking, mitochondrial fusion, and phagocytosis. Nevertheless, the function of the Vps13 protein in plant pathogenic fungi has not been explored. Here, we analysed the biological functions of the Vps13 protein in the development and pathogenicity of M. oryzae. Deletion mutants of MoVps13 significantly reduced the conidiation and decreased the rate of fungal infection on hosts. Moreover, the loss of MoVps13 resulted in defective cell wall integrity (CWI) and plasma membrane (PM) homeostasis when treated with chemicals for inducing cell wall stress (200 mg/mL Congo Red or 0.005% SDS) and sphingolipid synthesis inhibitors (2 μM myriocin or 2 μM amphotericin B). This indicated that MoVps13 is also involved in cell wall synthesis and sphingolipid synthesis. Through immunoblotting, autophagic flux detection, co-localization, and chemical drug sensitivity assays, we confirmed the involvement of Movps13 in ER-phagy and the response to ER stress. Additionally, we generated the C-terminal structure of MoVps13 with high accuracy using the alphaflod2 database. Our experimental evidence indicates that MoVps13 is an important virulence factor that regulates the pathogenicity of M. oryzae by controlling CWI, lipid metabolism and the ER-phagy pathway. These results have expanded our knowledge about pathogenic fungi and will help exploration for novel therapeutic strategies against the rice blast fungus.

Genetic Introgression Between Different Groups Reveals the Differential Process of Asian cultivated Rice

Genetic introgression plays an important role in the domestication of crops. The Asian cultivate rice consists of two major subspecies, they are indica and japonica. There are already many reports about existence of genetic introgression between the two subspecies. However, those studies often use few limited markers to characterize the genetic introgression that exists in some specific small populations. In this study we use the genome wide variation data of Asia cultivated rice to investigate their genetic introgression on the whole genome level. We detect a total of 13 significantly high introgression loci between the tropical japonica and indica population. Two different methods are used to identify the genetic introgression regions. For most of the detected introgression regions they generally get consistent results. Some previous known introgression genes are detected in the identified introgression loci, such as heat resistance gene TT1 and GLW7. The biological functions for these genetic introgression regions are annotated by the published QTL mapping results. We find that genetic introgression plays an important role in both the determination of the phenotype and the domestication process of different groups. Our study also provides useful information and resources for the study of rice gene function and domestication process.