Roger W. Innes

WALKING IN THE FOOTSTEPS OF HIS FAMILY

Innes spent his childhood in southern California, not far from the University of California, Riverside. The youngest of four sons born to parents with advanced science degrees, for Innes, pursuing a Ph.D. was always a given. Following in the footsteps of their father who had a doctorate in chemistry, two of Innes’ brothers received PhD degrees, one in physical chemistry and the other in particle physics, and his third brother received a Master’s in environmental engineering. It was his love for the environment and animals however, that directed Innes toward biology. As a high school student, Innes liked to spend a majority of his time outdoors. It wasn’t surprising therefore when he initially chose ecology for his undergraduate studies because of its lurking promise of travels to remote destinations and the study of exotic animals. During his degree at the Humboldt State University, however, this self-termed “naïve feeling” was displaced by his genuine interest in genetics and microbiology. Still, his wildlife biology major influenced his choice to pursue a PhD studying organismal interactions. Guided by the naturalist within, Innes moved to the University of Colorado in Boulder to pursue his doctoral studies in molecular microbiology against the backdrop of the Rocky Mountains.

A THOROUGH FOUNDATION IN PLANT–MICROBE INTERACTION STUDIES

The nomadic lifestyle of an early career scientist has its drawbacks, but it also offers some rewards. It exposes one to many interesting research topics while at the same time stimulating collaborations that advance scientific progress. As the only graduate student studying host–rhizobia interactions in the lab of Professor Peter L. Kuempel, Innes was offered the chance to perform research in the lab of a collaborator, Dr. Barry Rolfe, at Australian National University. During a five-month visit, Innes was able to show that plant factors secreted from the roots of clover plants induced expression of genes on the symbiosis plasmid of Rhizobium trifolii. In fact, his doctoral research contributed to the seminal discovery of flavones as inducers of Nod genes during root nodule symbiosis (Redmond et al., 1986).

Having received training in bacterial genetics, Innes next wanted to learn more about their interacting partners: plants. His search for a postdoc led him to his scientific mentor whom he regards as his most inspiring role model to date. Innes met visiting seminar speaker Brian Staskawicz at a round-table discussion with graduate students at the University of Colorado. Innes remembers recognizing Brian as a clear leader in his field even then, having just cloned the first avirulence (avr) gene from Pseudomonas syringae. He enjoyed their brief conversation enough to apply for an National Science Foundation fellowship in plant molecular biology to conduct post-doctoral research in the Staskawicz lab at the University of California, Berkeley. Here Innes was involved in three projects; his primary project pioneered the use of Arabidopsis (Arabidopsis thaliana) as a model for plant pathogen interactions. With the interest in Arabidopsis as a genetic model rising, Innes started by establishing a transposon tagging system in Arabidopsis using the Ac/Ds transposon system of maize. On the second project, he cloned the avrRpt2 gene of P. syringae (Whalen et al., 1991). In his third project, he showed that an avr gene recognized by soybean (Glycine max; avrB) was also recognized by Arabidopsis, which he then developed into a full-fledged research program for starting his own lab at Indiana University in 1991, after only three years of postdoctoral research (Innes et al., 1993a, 1993b).

RESARCH ON NLR RESISTANCE GENES AND THE “MOUSETRAP” MODEL

Plant survival depends on the inherent or .innate immune system that is activated upon detection of pathogens. NLR proteins that confer disease resistance are active components of this robust immune system. NLRs can either directly recognize and bind pathogen effector proteins or indirectly recognize host proteins modified by these effectors (Qi and Innes, 2013). Innes’ contribution to what we know today about NLRs is tremendous.

In 1991, the concept of an immune system in plants was just emerging and it was yet unknown what a resistance or “R” gene encoded. What was known, however, was that these R genes recognized matching or cognate effector proteins, also called Avirulence proteins. As a National Science Foundation postdoctoral fellow, Innes used the avrB gene from the soybean pathogen Pseudomonas syringae pv glycinea to screen for disease resistance across 53 Arabidopsis ecotypes. The premise of the screen was straightforward: if AvrB were recognized, Arabidopsis leaves would trigger a hypersensitive response (HR) marked by the development of dead cells containing lesions. If however, no matching R gene was present in the ecotype, the effector would help suppress the immune system leading to development of disease. Innes identified multiple ecotypes of Arabidopsis that induced an HR in response to AvrB, and then showed that this response was mediated by a single dominant locus RESISTANCE TO PSEUDOMONAS SYRINGAE3 (RPS3) present only in the resistant ecotypes of Arabidopsis (Innes et al., 1993b). As an early career project leader, Innes and his group further determined that Arabidopsis RPS3 mapped to the same location as RPM1, a disease resistance gene being characterized by Jeff Dangl’s laboratory based on its ability to recognize an unrelated avr gene, avrRpm1, and that mutation of RPS3 abolished RPM1 function, establishing that a single plant disease resistance gene could mediate recognition of two unrelated effectors (Bisgrove et al., 1994). One year later, together with Dangl’s group, Innes’ lab elucidated that RPM1/RPS3 encoded an NLR protein, which represented just the third NLR gene cloned, following the N gene of tobacco (Nicotiana tabacum) and the RPS2 gene from Arabidopsis (Grant et al., 1995).

When asked what he thinks is his biggest contribution to plant science today, Innes returns to the mousetrap model. In their proof of concept study published in the journal Science, Innes and colleagues showed that changing six amino acids of the intermediary “bait” protein kinase PBS1 could turn it into a target for both bacterial and viral pathogens that used proteases for infection. Once cleaved by the microbial protease, the associated NLR protein (trap) RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5) triggered a cell death response that limited disease spread. Since many pathogens including fungi and oomycetes use proteases for infectivity, the potential impact of this finding is large. PBS1 is highly conserved across all flowering plants, and AvrPphB induces an HR in most crop species, including soybean and wheat (Triticum aestivum; Russell et al., 2015; Carter et al., 2018); therefore, it should be possible to engineer PBS1 “decoys” that will confer resistance to economically important pathogens in most crops.

MAKING ADVANCES THROUGH MENTORING AND TEACHING

Innes reveals that he has always led a relatively small lab with a mix of postdoctoral fellows and graduate students. Their work however, has sometimes led him in unexpected directions and opened new avenues of research.

He recalls a screen initiated by his first postdoc Catherine Frye, who identified six Arabidopsis mutants resistant to the bacterial pathogen P. syringae DC3000. When Innes and Frye infected the six P. syringae resistant Arabidopsis plants with the powdery mildew fungus Golovinomyces cichoracearum, a single mutant line exhibited resistance to both the bacteria and the fungus! On this mutant, pathogenic spores germinated and formed hyphae normally, but could not develop conidiophores because of excessive cell death. This late-acting gene that conferred a broad spectrum disease resistance was named ENHANCED DISEASE RESISTANCE1 (EDR1; Frye and Innes, 1998). Subsequently they established that it encoded a protein kinase that is conserved across flowering plants, and that it functions as a key negative regulator of cell death during both biotic and abiotic stress responses (Frye et al., 2001; Tang et al., 2005). Frye’s discoveries led Innes to adopt powdery mildew as a model fungal pathogen in the lab, and to adopt cell biology as an essential approach to understanding plant:pathogen interactions. More recently, Innes’ venture into the field of extracellular vesicles, described below, was initiated by a single graduate student, Brian Rutter.

In addition to being a respected mentor, Innes is an award-winning teacher at Indiana University, where he currently teaches a genetics laboratory course. His teaching philosophy is simple: get students engaged. Innes believes that inquiry-based learning and not lecture-based teaching ensures effective communication. As an example, students in his course are charged with finding current popular press articles on genetics topics that summarize recent peer-reviewed research papers. For extra credit, students are asked to compare and contrast the research papers to their corresponding popular press articles. In this way, not only do students actively follow and critically evaluate pioneering research, they also gain an understanding of how science impacts society. Each week, Innes discusses a student submission with the entire class that is relevant to that week’s lab activities. For example, CRISPR-based editing of human embryos generated a memorable discussion before the students themselves embarked on a genome editing project of their own!

“THE FUTURE IS ABSOLUTELY—CELL BIOLOGY!”

Innes is especially excited while talking about plans for his future research. “The future is absolutely cell biology!” he exclaims. “While using electron microscopy to better understand how plant cells die during the hypersensitive response (HR), we stumbled upon extracellular vesicles (EVs) that appeared to be functioning in intercellular communication.” But what did these EVs contain and could they be purified? In their recent breakthrough paper, Rutter and Innes described a method to isolate intact EVs from apoplastic fluids of Arabidopsis (Rutter and Innes, 2017). The concentration of EVs in apoplastic fluid doubled when plants were treated with salicylic acid or infected with the P. syringae, suggesting they play a central role in plant immunity. Innes’ lab has recently teamed up with the laboratory of fellow Plant Cell editor Blake Meyers to analyze the RNA content of EVs, discovering that they are highly enriched in a previously overlooked class of small RNAs that are only 10-17 nucleotides in length, which they have dubbed “tiny RNAs” (tyRNAs; Baldrich et al., 2019). With this line of research, Innes hopes to answer fundamental questions regarding how intercellular communication is accomplished in plants, and how plants and microbes communicate. “Answering these questions will be key to harnessing EVs for applications in agriculture, and possibly even medicine,” Innes states, “and fortunately, I have a great team of graduate students eager to answer them.”