In Planta Study Localizes an Effector Candidate from Austropuccinia psidii Strain MF-1 to the Nucleus and Demonstrates In Vitro Cuticular Wax-Dependent Differential Expression.

Austropuccinia psidii is a biotrophic fungus that causes myrtle rust. First described in Brazil, it has since spread to become a globally important pathogen that infects more than 480 myrtaceous species. One of the most important commercial crops affected by A. psidii is eucalypt, a widely grown forestry tree. The A. psidii-Eucalyptus spp. interaction is poorly understood, but pathogenesis is likely driven by pathogen-secreted effector molecules. Here, we identified and characterized a total of 255 virulence effector candidates using a genome assembly of A. psidii strain MF-1, which was recovered from Eucalyptus grandis in Brazil. We show that the expression of seven effector candidate genes is modulated by cell wax from leaves sourced from resistant and susceptible hosts. Two effector candidates with different subcellular localization predictions, and with specific gene expression profiles, were transiently expressed with GFP-fusions in Nicotiana benthamiana leaves. Interestingly, we observed the accumulation of an effector candidate, Ap28303, which was upregulated under cell wax from rust susceptible E. grandis and described as a peptidase inhibitor I9 domain-containing protein in the nucleus. This was in accordance with in silico analyses. Few studies have characterized nuclear effectors. Our findings open new perspectives on the study of A. psidii-Eucalyptus interactions by providing a potential entry point to understand how the pathogen manipulates its hosts in modulating physiology, structure, or function with effector proteins.

The key role of indole-3-acetic acid biosynthesis by Bacillus thuringiensis RZ2MS9 in promoting maize growth revealed by the ipdC gene knockout mediated by the CRISPR-Cas9 system.

The bacterial biosynthesis of indole-3-acetic acid (IAA) is often related to the beneficial effects of plant growth-promoting rhizobacteria (PGPR) on plant development. In PGPR belonging to the Bacillus genus, the synthesis of IAA may occur through different metabolic pathways that are still poorly understood. B. thuringiensis (Bt) is well known for its insecticidal properties; however, its beneficial features are not limited to pest control. Our group has been studed the beneficial effects of Bt strain RZ2MS9 as growth promoter in a range of plant crops, including soybean, tomato, and maize. We recently demonstrated that bacterial IAA biosynthesis plays an important role in the ability of RZ2MS9 to benefit plant development. However, the molecular involved mechanisms in the IAA biosynthesis by this bacterium in the beneficial interaction with plants remain unclear. Here, we investigated the genetic basis of IAA biosynthesis by RZ2MS9. We knocked out the ipdC gene, involved in IAA biosynthesis via the tryptophan-dependent IPyA pathway, using the CRISPR-Cas9 system. Our results showed that, by disrupting the IPyA pathway, the amount of IAA synthesized by the mutant RZ2MS9 (ΔipdC) in the presence of tryptophan drops 57%. The gene knockout did not affect the bacterial growth, but it did affect its ability to colonize maize. Moreover, deactivating the ipdC gene in RZ2MS9 significantly reduces its ability to promote maize growth. ΔipdC performed worse than RZ2MS9 in almost all evaluated plant parameters, including total root length, projected root area, lateral roots, aerial part dry matter, and germination speed index. Therefore, we demonstrated that tryptophan-dependent IAA biosynthesis via the IPyA pathway by RZ2MS9 is strongly influenced by the ipdC gene. Furthermore, IAA biosynthesis by RZ2MS9 is a major mechanism used by this PGPR to promote maize growth.

The auxin-producing Bacillus thuringiensis RZ2MS9 promotes the growth and modifies the root architecture of tomato (Solanum lycopersicum cv. Micro-Tom).

Strains of Bacillus thuringiensis (Bt) are commonly commercialized as bioinoculants for insect pest control, but their benefits go beyond their insecticidal property: they can act as plant growth-promoters. Auxins play a major role in the plant growth promotion. However, the mechanism of auxin production by the Bacilli group, and more specifically by Bt strains, is unclear. In previous work, the plant growth-promoting rhizobacterium (PGPR) B. thuringiensis strain RZ2MS9 increased the corn roots. This drew our attention to the strain's auxin production trait, earlier detected in vitro. Here, we demonstrate that in its genome, RZ2MS9 harbours the complete set of genes required in two pathways that are used for Indole acetic acid (IAA) production. We also detected that the strain produces almost five times more IAA during the stationary phase. The bacterial application increased the shoot dry weight of the Micro-Tom (MT) tomato by 24%. The application also modified MT root architecture, with an increase of 26% in the average lateral root length and inhibition of the axial root. At the cellular level, RZ2MS9-treated MT plants presented elongated root cortical cells with intensified mitotic activity. Altogether, these are the best characterized auxin-associated phenotypes. Besides that, no growth alteration was detected in the auxin-insensitive diageotropic (dgt) plants either with or without the RZ2MS9 inoculation. Our results suggest that auxins play an important role in the ability of B. thuringiensis RZ2MS9 to promote MT growth and provide a better understanding of the auxin production mechanism by a Bt strain.

Gene Knockout of Beneficial Plant-associated Bacillus spp. Using the CRISPR-Cas9 Double Plasmid System.

Bacillus spp. have great agricultural potential as a plant growth promoter and biocontrol agent. However, little is known concerning the bacterial molecular basis for the improvement of plant fitness. Thus, it is highly desirable to develop techniques that can contribute to the elucidation of the genetic basis for the mechanisms involved in beneficial bacterium-plant interactions. In this context, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 is a powerful tool based on programmable molecular scissors that perform precise incisions in any DNA sequence. CRISPR-Cas9 can alter gene sequences and constitutes a cutting-edge tool to elucidate the role and function of bacterial genes associated with the benefits of plant interactions. The method described here uses a feasible CRISPR-Cas9 system in a double plasmid, one plasmid harboring the Cas9 endonuclease and the other the sgRNA, to promote gene knockout/editing in the Bacillus genus. This approach favors high efficiency in generating mutants for one or more genes in continuous or multiplex editing. Additionally, due to its universality, it can be applied to genera other than Bacillus.

The Eucalyptus Cuticular Waxes Contribute in Preformed Defense Against Austropuccinia psidii.

Austropuccinia psidii, the causal agent of myrtle rust, is a biotrophic pathogen whose growth and development depends on the host tissues. The uredospores of A. psidii infect Eucalyptus by engaging in close contact with the host surface and interacting with the leaf cuticle that provides important chemical and physical signals to trigger the infection process. In this study, the cuticular waxes of Eucalyptus spp. were analyzed to determine their composition or structure and correlation with susceptibility/resistance to A. psidii. Twenty-one Eucalyptus spp. in the field were classified as resistant or susceptible. The resistance/susceptibility level of six Eucalyptus spp. were validated in controlled conditions using qPCR, revealing that the pathogen can germinate on the eucalyptus surface of some species without multiplying in the host. CG-TOF-MS analysis detected 26 compounds in the Eucalyptus spp. cuticle and led to the discovery of the role of hexadecanoic acid in the susceptibility of Eucalyptus grandis and Eucalyptus phaeotricha to A. psidii. We characterized the epicuticular wax morphology of the six previously selected Eucalyptus spp. using scanning electron microscopy and observed different behavior in A. psidii germination during host infection. It was found a correlation of epicuticular morphology on the resistance to A. psidii. However, in this study, we provide the first report of considerable interspecific variation in Eucalyptus spp. on the susceptibility to A. psidii and its correlation with cuticular waxes chemical compounds that seem to play a synergistic role as a preformed defense mechanism.