Calmodulin Binds MLO Protein in Barley to Confer Mildew Resistance – Recent Breakthrough in Plant Defense Mechanism

Although mlo mutants in barley were reported to show stable and broad-spectrum resistance to powdery mildew more than sixty years ago, it is only recently that the mode of operation of the mutant gene has begun to be understood. In a review article in the August issue of Trends in Plant Science (2002) vol 7:379-380, Stein Monica and Shauna C. Somerville at Carnegie Institution, Dept. of Plant Biology, Stanford CA, have discussed the current status of our knowledge about the functioning of this recessive gene.

Barley powdery mildew is caused by the pathogen Blumeria graminis f. sp. hordei. This pathogen is biotrophic because it derives nutrition from the host without killing it. The authors describe how the mlo individuals counter the attack of pathogens. Compared to the wild type (Mlo-susceptible lines), penetration of fungal hyphae (~50–70%) drop from 50-70 to below 1% on mlo-resistant lines. Death of mesophyll cells occurs at the later stage of infection. Following pathogen attack, mlo plants have been shown to build up defense-related markers, besides accumulating p-coumaroyl- hydroxyagmatine in a preferential manner.

Similarity of structure between GCRP and the MLO protein: The structure of the Mlo gene-encoded protein and that of G-coupled receptor protein (GCRP) is similar. MLO is an average sized protein and its homologues are found only in plants. Both MLO and GCRP proteins are transmembrane proteins, composed of a single protein chain that crosses the cell membrane seven times. A study to find out the inter-relationship between the MLO- and GCRP revealed that the resemblance between the two may be superficial. Bombardment of both resistant and susceptible barley plants with various G subunits of the heterotrimeric G-protein complex did not bring about any alteration in their response to pathogen nor was there any observed difference between the Arabidopsis G mutant and a wild-type control in their susceptibility to powdery mildew. The conclusion was that heterotrimeric G-proteins do not play any significant role in MLO suppression of disease resistance.

Role of RAC in mildew resistance scenario: Two more interesting findings regarding the role of the RAC family of proteins in conferring mildew resistance came to light. RACs are monomeric Rho-type GTPases. They take part in signaling and programmed cell death. Results of a recent study showed that there was a partial restoration of powdery mildew resistance in an otherwise susceptible Mlo barley variety as a result of inactivation of its RacB gene by RNAi (i = interference).

Another interesting piece of data in this connection was that mlo mutants lose their levels of resistance partially when mutation occurs at another locus, Ror1. A clear cut answer to the question whether RACB plays a direct role in MLO suppression of plant defenses and cell death and whether the two genes act in the same or two different pathways remains unsolved.

Experiments with single epidermal cells to study mlo resistance: Results of an ingenious experiment in which single epidermal cells were subjected to biolistic bombardment, reveal that mlo resistance is only effective in cells where a functional MLO is absent. Levels of suscepti- bility to powdery mildew infection in plants overexpressing Mlo increased to a great extent, indicating that MLO acts as a negative regulator, thereby facilitating infection. Further confirmation that MLO is a negative regulator was obtained from the observation that its loss puts into action plant defense mechanism in uninfected tissues.

Does MLO play a general role in limiting cell death? Another interesting observation is that in the mutant mlo, senescence proceeds faster than in the wild type, even though the process may initiate at the same time in the two sets of plants. These data suggest that MLO is involved in orchestrating the cell death process. According to the authors, enhanced levels of Mlo mRNA following stress may be due to the production of toxic reactive oxygen species

MLO as a negative regulator: Results of recent experiments seem to suggest that in senescent-, stressed- and pathogen-invaded plants, MLO acts as a general negative regulator of cell death. Although the resistance to mildew attack is robust in mlo plants, the latter are more likely to die in response to other stresses. The above assumption does not explain as to why the progression of powdery mildew infection halts at the penetration stage, much ahead of cell death. There is no explanation also as to why mlo resistance does not act against a number of other pathogens, sharing characteristics of powdery mildew.

Recent findings to show that the binding of MLO to calmodulin enhances resistance: The authors bring into focus two recent findings, shedding light on how MLO may act in the defense mechanism. One was the identification of a small region of the C-terminal domain of MLO binding to calmodulin. The characteri- zation study of this region revealed that replacement of single amino acids knocks off barley MLO-calmodulin interaction. Further- more, in vitro experiments, it was shown that levels of resistance were increased in mutants carrying the MLO protein in which the above region did not bind calmodulin. Increased resistance as compared to Wild-type indicated that calmodulin-MLO binding is one of the essential factors for MLO to be fully functional. Participation of calmodulin in MLO function suggests that intracellular Ca++ anions binding to calmodulin can modulate plant defenses and cell death.

Participation of a small G protein enhances levels of resistance: The second important finding was the discovery of a small G protein, RacB. This small G-protein has been implicated in mlo resistance, unlike heterotrimeric G-proteins which were earlier shown to have no participatory role in defense. The authors point out that MLO has 15 homologs in Arabidopsis. Consequently, characterizing only MLO protein with a defined role is not sufficient to uncover defense mechanism. Any future program should therefore include characterization of all the 15 MLO homologs.

Although a lot of ground has been covered during the past sixty years or so, there is still a long way to go before all the puzzle pieces are put together to obtain a full picture of mlo resistance. The technological innovations and the unraveling of the entire base sequences of Arabidopsis and rice genomes together with the recent findings of the role of calmodulin and a monomeric G-protein RacB in mlo resistance, brighten the prospect towards attainment of that objective.

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