The effects of endophytic Fusarium verticillioides on the interactions of maize and its fungal pathogen Ustilago maydis.

University of Minnesota Ph.D. dissertation. August 2010. Major: Plant Biological Sciences. Advisor: Goergiana May, Ph.D. 1 computer file (PDF); xi, 113 pages.Diverse microbial organisms, including mycorrhizal fungi, endophytes and pathogens inhabit plants, interact with each other, and affect their fitness. Although theoretical studies suggest that the outcomes of multispecies interactions are often different from those of pairwise interactions, most empirical studies have focused on pairwise plant-pathogen interactions. Using endophytic isolates of Fusarium verticillioides (Sacc.) Nirenberg, the corn smut pathogen, Ustilago maydis DC (Corda) and maize, our studies suggest that endophytes could play important ecological roles for host defense and their impact needs to be appreciated when studying plant interactions with other organisms occurring in the same host. First, our results suggest that F. verticillioides likely interacts with U. maydis directly to reduce the host damage by pathogen infections, which we define here as 'aggressiveness.' Since the endophyte alone did not have detectable effects on plant growth, we inferred that F. verticillioides indirectly improves plant growth in the presence of the pathogen, U. maydis . Secondly, we found that U. maydis aggressiveness is constrained by the genetic association between traits governing aggressiveness and fitness, i.e., trade-off, and the endophyte, F. verticillioides enforces limits to U. maydis aggressiveness. Pathogen fitness decreases as the level of aggressiveness increases. Surprisingly, endophyte co-inoculation with the pathogen resulted in increased pathogen fitness, likely because the biotrophic pathogen, U. maydis depends on plant resources for its reproduction and plants in the endophyte co-inoculation treatments grow better than do plants in the pathogen only inoculation treatments. Lastly, we found strain-specific effects of the endophyte on the ecological and fitness outcomes of maize- U. maydis interactions. The endophyte strain which produced least amount of fusaric acid had least impact on U. maydis aggressiveness, suggesting that the secreted secondary compound of the endophyte may play antagonistic role against the pathogen. Together, these results suggest that F. verticillioides endophytes play important defensive roles for host plants and that the evolution of plant-pathogen interactions is responsive to the microbial environment in which they occur

A Genome-Wide Survey of Highly Expressed Non-Coding RNAs and Biological Validation of Selected Candidates in Agrobacterium tumefaciens

Agrobacterium tumefaciens is a plant pathogen that has the natural ability of delivering and integrating a piece of its own DNA into plant genome. Although bacterial non-coding RNAs (ncRNAs) have been shown to regulate various biological processes including virulence, we have limited knowledge of how Agrobacterium ncRNAs regulate this unique inter-Kingdom gene transfer. Using whole transcriptome sequencing and an ncRNA search algorithm developed for this work, we identified 475 highly expressed candidate ncRNAs from A. tumefaciens C58, including 101 trans-encoded small RNAs (sRNAs), 354 antisense RNAs (asRNAs), 20 5′ untranslated region (UTR) leaders including a RNA thermosensor and 6 riboswitches. Moreover, transcription start site (TSS) mapping analysis revealed that about 51% of the mapped mRNAs have 5′ UTRs longer than 60 nt, suggesting that numerous cis-acting regulatory elements might be encoded in the A. tumefaciens genome. Eighteen asRNAs were found on the complementary strands of virA, virB, virC, virD, and virE operons. Fifteen ncRNAs were induced and 7 were suppressed by the Agrobacterium virulence (vir) gene inducer acetosyringone (AS), a phenolic compound secreted by the plants. Interestingly, fourteen of the AS-induced ncRNAs have putative vir box sequences in the upstream regions. We experimentally validated expression of 36 ncRNAs using Northern blot and Rapid Amplification of cDNA Ends analyses. We show functional relevance of two 5′ UTR elements: a RNA thermonsensor (C1_109596F) that may regulate translation of the major cold shock protein cspA, and a thi-box riboswitch (C1_2541934R) that may transcriptionally regulate a thiamine biosynthesis operon, thiCOGG. Further studies on ncRNAs functions in this bacterium may provide insights and strategies that can be used to better manage pathogenic bacteria for plants and to improve Agrobacterum-mediated plant transformation.This is an article from PLoS One 8 (2013): e70720, doi: 10.1371/journal.pone.0070720. Posted with permission.</p

Identifying Factors that Determine Effectiveness of Delivery Agents in Biolistic Delivery Using a Library of Amine-Containing Molecules

Biolistic transfection is a popular and versatile tool for plant transformation. A key step in the biolistic process is the binding of DNA to the heavy microprojectile using a delivery agent, usually a positively charged molecule containing amine groups. Currently, the choice of the commercial delivery agent is mostly limited to spermidine. In addition, the detailed delivery mechanism has not been reported. To help broaden the selection of the delivery agent and reveal the fundamental mechanisms that lead to high delivery performance, a library of amine-containing molecules was investigated. A double-barrel biolistic delivery device was utilized for testing hundreds of samples with much-improved consistency. The performance was evaluated on onion epidermis. The binding and release of DNA were measured via direct high-performance liquid chromatography analysis. This study shows that the overwhelming majority of the amine library performed at the same level as spermidine. To further interpret these results, correlations were performed with thousands of molecular descriptors generated by chemical modeling. It was discovered that the overall charge is most likely the key factor to a successful binding and delivery. Furthermore, even after increasing the amount of the DNA concentration 50-fold to stress the binding capacity of the molecules, the amines in the library continued to deliver at a near identical level while binding all the DNA. The increased DNA was also demonstrated with a Cas9 editing test that required a large amount of DNA to be delivered, and the result was consistent with the previously determined amine performance. This study greatly expands the delivery agent selection for biolistic delivery, allowing alternatives to a commercial reagent that are more shelf-stable and cheaper. The library also offers an approach to investigate more challenging delivery of protein and CRISPR-Cas via the biolistic process in the future.This document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in ACS Applied Bio Materials, copyright © 2022 American Chemical Society after peer review. To access the final edited and published work see DOI: 10.1021/acsabm.2c00689. Posted with permission

Distribution of ncRNAs on four replicons.

<p>Distribution of ncRNAs on four replicons.</p

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種別: 実習報

Validated ncRNAs with Northern blot analysis.

*<p>ncRNAs have been validated with 5′ and 3′ RACE.</p>†<p>ncRNAs have been previously identified or detected by Wilms et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070720#pone.0070720-Wilms3" target="_blank">[51]</a>.</p><p>F and R at the end of each ncRNA tag denote strand information: Forward and Reverse.</p

Transcriptional regulation of <i>thiCOGG</i> operon by a TPP riboswitch (C1_2541934R).

<p>A putative riboswitch at the 5′ UTR of thiamine biosynthesis operon, <i>thiCOGG</i>, transcriptionally regulates gene expression. <b>A</b>) Secondary structure predicted by mFold web server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070720#pone.0070720-Zuker1" target="_blank">[94]</a>: Δ<i>G</i>  = −35.08 kcal/mol. (<b>B</b>) Northern blot analysis with a probe specific to the riboswitch and (<b>C</b>) a probe specific to the downstream gene, <i>thiC</i> (<b>C</b>). Total RNA was isolated from <i>A. tumefaciens</i> strain C58 grown in YEP medium until mid-log phase (YEP-L), YEP medium until late stationary phase (YEP-S), AB induction medium without AS (AB), AB induction medium with AS (IND), and AB with 100 µg/mL of thiamine (AB+Thi). +RP, RNA samples were treated with RNAprotect Bacteria reagent (Qiagen, USA). *AB and *AB+Thi, RNA samples were not treated. Ethidiumbromide stained 16S rRNA bands were included as loading control.</p

Variation of 5′ UTR length.

<p>The distance between TSS and start codon (5′ UTR) varied substantially from 0 to 521 nt, averaging 88 nt and median of 61 nt. Among the 675 protein coding genes, 1.8% (12) were leaderless (≤10 nt), 39% (253) were short (11∼50 nt), while 30% (203) were long (>100 nt). About 51% (345) of 5′ UTRs were longer than 60 nt.</p

Validation of selected ncRNAs by Northern blot analysis.

<p>Depth of coverage profiles and Northern hybridization images of 22 <i>Agrobacterium</i> ncRNAs under four growth conditions: YEP medium until mid-log phase (YEP-L), YEP medium until late stationary phase (YEP-S), AB induction medium without AS (AB), AB induction medium with AS (IND). (<b>A</b>) Fifteen ncRNAs encoded on the circular chromosome (C1), (<b>B</b>) five ncRNAs encoded on the linear chromosome (C2), and (<b>C</b>) two ncRNAs encoded on the pAt plasmid (pAt).</p

Induction of <i>vir</i> genes with AS.

<p>Expression of 24 <i>vir</i> genes with and without AS was visualized for data validation. Depth of coverage at each nucleotide position from 180,590 to 211,094 of Ti plasmid was plotted for (<b>A</b>) forward strand and (<b>B</b>) reverse strand. A total of 24 <i>vir</i> genes were included: <i>virA</i>, <i>virB</i> (B1∼B11), <i>virG</i>, <i>virC</i> (C1, C2), <i>virD</i> (D1∼D5) and <i>virE</i> (E0∼E3).</p