The ectomycorrhizal fungus Paxillus ammoniavirescens influences the effects of salinity on loblolly pine in response to potassium availability.

Salinity is an increasing problem in coastal areas affected by saltwater intrusion, with deleterious effects on tree health and forest growth. Ectomycorrhizal (ECM) fungi may improve the salinity tolerance of host trees, but the impact of external potassium (K+ ) availability on these effects is still unclear. Here, we performed several experiments with the ECM fungus Paxillus ammoniavirescens and loblolly pine (Pinus taeda L.) in axenic and symbiotic conditions at limited or sufficient K+ and increasing sodium (Na+ ) concentrations. Growth rate, biomass, nutrient content, and K+ transporter expression levels were recorded for the fungus, and the colonization rate, root development parameters, biomass, and shoot nutrient accumulation were determined for mycorrhizal and non-mycorrhizal plants. P. ammoniavirescens was tolerant to high salinity, although growth and nutrient concentrations varied with K+ availability and increasing Na+ exposure. While loblolly pine root growth and development decreased with increasing salinity, ECM colonization was unaffected by pine response to salinity. The mycorrhizal influence on loblolly pine salinity response was strongly dependent on external K+ availability. This study reveals that P. ammoniavirescens can reduce Na+ accumulation of salt-exposed loblolly pine, but this effect depends on external K+ availability.

Agrobacterium tumefaciens-mediated transformation of Nigrospora sp. isolated from switchgrass leaves and antagonistic toward plant pathogens.

Nigrospora is a diverse genus of fungi colonizing plants through endophytic, pathogenic, or saprobic interactions. Endophytic isolates can improve growth and development of host plants, as well as their resistance to microbial pathogens, but exactly how they do so remains poorly understood. Developing a reliable transformation method is crucial to investigate these mechanisms, in particular to identify pivotal genes for specific functions that correlate with specific traits. In this study, we identified eight isolates of Nigrospora sp. internally colonizing the leaves of switchgrass plants cultivated in North Carolina. Using an Agrobacterium tumefaciens-mediated transformation approach with control and GFP-expressing vectors, we report the first successful transformation of two Nigrospora isolates. Finally, we demonstrate that wild-type and transgenic isolates both negatively impact the growth of two plant pathogens in co-culture conditions, Bipolaris maydis and Parastagonospora nodorum, responsible for the Southern Leaf Blight and Septoria Nodorum Blotch diseases, respectively. The GFP-transformed strains developed here can therefore serve as accurate reporters of spatial interactions in future studies of Nigrospora and pathogens in the plant. Finally, the transformation method we describe lays the foundation for further genetic research on the Nigrospora genus to expand our mechanistic understanding of plant-endophyte interactions.

Development of a rapid RNA extraction procedure from urediniospores of the leaf rust fungus, Puccinia triticina.

Obtaining high quality RNA in good quantities is often a requirement for plant-pathogen interaction studies, so it becomes very essential that a highly efficient method should be deployed to isolate RNA from minute quantities of fungal spores. The methods available to date, either require a high quantity of spores or the use of expensive chemicals. The protocol discussed here for RNA isolation from Puccinia triticina pathotype 77-5 urediniospores utilizes TRI Reagent as extraction buffer that is widely used for RNA isolation from plant tissues. Urediniospores have a tough cell wall as compared to other plant cells. Therefore, the protocol was optimized keeping the primary focus on quickly disrupting cell walls. Two different methods, one using a combination of liquid nitrogen and ultrasonic water-bath and the other method using micro-homogenizer were utilized for crushing the spores in the present study. The developed methods do not utilize mortar and pestle, instead they promote direct crushing of urediniospores in tubes; thereby minimizing sample loss and enhancing quality.

Leaf rust (Puccinia triticina) mediated RNAi in wheat (Triticum aestivum L.) prompting host susceptibility.

Significance of microRNAs in regulating gene expression in higher eukaryotes as well as in pathogens like fungi to suppress host defense is a well-established phenomenon. The present study focuses on leaf rust fungi Puccinia triticina (Pathotype 77-5) mediated RNAi to make wheat (Triticum aestivum L.) more susceptible. To reach such conclusions, we first confirmed the presence of argonaute (AGO) and dicer-like protein (DCL) family sequences in Puccinia. Bioinformatic tools were applied to retrieve the sequences from Puccinia genome followed by cloning and sequencing from P. triticina pathotype 77-5 cDNA to obtain the specific sequences. Their homologs were searched in other 14 Puccinia races to relate them with pathogenesis. Further, precursor sequences for three miRNA-like RNA molecules (milRs) were cloned from P. triticina cDNA. Their target genes like MAP kinase were successfully predicted and validated through degradome mapping and qRT-PCR. Gradual increase in milR2 (milR and milR*) expression over progressive time point of infection and positive expression for all the milRs within 77-5 urediniospores confirmed a complete host- independent RNAi activity by P. triticina.

Identification and molecular characterization of a trans-acting small interfering RNA producing locus regulating leaf rust responsive gene expression in wheat (Triticum aestivum L.).

MAIN CONCLUSION: A novel leaf rust responsive ta-siRNA-producing locus was identified in wheat showing similarity to 28S rRNA and generated four differentially expressing ta-siRNAs by phasing which targeted stress responsive genes. Trans-acting-small interfering RNAs (Ta-siRNAs) are plant specific molecules generally involved in development and are also stress responsive. Ta-siRNAs identified in wheat till date are all responsive to abiotic stress only. Wheat cultivation is severely affected by rusts and leaf rust particularly affects grain filling. This study reports a novel ta-siRNA producing locus (TAS) in wheat which is a segment of 28S ribosomal RNA but shows differential expression during leaf rust infestation. Four small RNA libraries prepared from wheat Near Isogenic Lines were treated with leaf rust pathogen and compared with untreated controls. A TAS with the ability to generate four ta-siRNAs by phasing events was identified along with the microRNA TamiR16 as the phase initiator. The targets of the ta-siRNAs included α-gliadin, leucine rich repeat, trans-membrane proteins, glutathione-S-transferase, and fatty acid desaturase among others, which are either stress responsive genes or are essential for normal growth and development of plants. Expression of the TAS, its generated ta-siRNAs, and their target genes were profiled at five different time points after pathogen inoculation of susceptible and resistant wheat isolines and compared with mock-inoculated controls. Comparative analysis of expression unveiled differential and reciprocal relationship as well as discrete patterns between susceptible and resistant isolines. The expression profiles of the target genes of the identified ta-siRNAs advocate more towards effector triggered susceptibility favouring pathogenesis. The study helps in discerning the functions of wheat genes regulated by ta-siRNAs in response to leaf rust.

Uncovering leaf rust responsive miRNAs in wheat (Triticum aestivum L.) using high-throughput sequencing and prediction of their targets through degradome analysis.

MAIN CONCLUSION: Deep sequencing identified 497 conserved and 559 novel miRNAs in wheat, while degradome analysis revealed 701 targets genes. QRT-PCR demonstrated differential expression of miRNAs during stages of leaf rust progression. Bread wheat (Triticum aestivum L.) is an important cereal food crop feeding 30 % of the world population. Major threat to wheat production is the rust epidemics. This study was targeted towards identification and functional characterizations of micro(mi)RNAs and their target genes in wheat in response to leaf rust ingression. High-throughput sequencing was used for transcriptome-wide identification of miRNAs and their expression profiling in retort to leaf rust using mock and pathogen-inoculated resistant and susceptible near-isogenic wheat plants. A total of 1056 mature miRNAs were identified, of which 497 miRNAs were conserved and 559 miRNAs were novel. The pathogen-inoculated resistant plants manifested more miRNAs compared with the pathogen infected susceptible plants. The miRNA counts increased in susceptible isoline due to leaf rust, conversely, the counts decreased in the resistant isoline in response to pathogenesis illustrating precise spatial tuning of miRNAs during compatible and incompatible interaction. Stem-loop quantitative real-time PCR was used to profile 10 highly differentially expressed miRNAs obtained from high-throughput sequencing data. The spatio-temporal profiling validated the differential expression of miRNAs between the isolines as well as in retort to pathogen infection. Degradome analysis provided 701 predicted target genes associated with defense response, signal transduction, development, metabolism, and transcriptional regulation. The obtained results indicate that wheat isolines employ diverse arrays of miRNAs that modulate their target genes during compatible and incompatible interaction. Our findings contribute to increase knowledge on roles of microRNA in wheat-leaf rust interactions and could help in rust resistance breeding programs.