Citrus MAF1, a repressor of RNA polymerase III, binds the Xanthomonas citri canker elicitor PthA4 and suppresses citrus canker development.

Transcription activator-like (TAL) effectors from Xanthomonas species pathogens act as transcription factors in plant cells; however, how TAL effectors activate host transcription is unknown. We found previously that TAL effectors of the citrus canker pathogen Xanthomonas citri, known as PthAs, bind the carboxyl-terminal domain of the sweet orange (Citrus sinensis) RNA polymerase II (Pol II) and inhibit the activity of CsCYP, a cyclophilin associated with the carboxyl-terminal domain of the citrus RNA Pol II that functions as a negative regulator of cell growth. Here, we show that PthA4 specifically interacted with the sweet orange MAF1 (CsMAF1) protein, an RNA polymerase III (Pol III) repressor that controls ribosome biogenesis and cell growth in yeast (Saccharomyces cerevisiae) and human. CsMAF1 bound the human RNA Pol III and rescued the yeast maf1 mutant by repressing tRNA(His) transcription. The expression of PthA4 in the maf1 mutant slightly restored tRNA(His) synthesis, indicating that PthA4 counteracts CsMAF1 activity. In addition, we show that sweet orange RNA interference plants with reduced CsMAF1 levels displayed a dramatic increase in tRNA transcription and a marked phenotype of cell proliferation during canker formation. Conversely, CsMAF1 overexpression was detrimental to seedling growth, inhibited tRNA synthesis, and attenuated canker development. Furthermore, we found that PthA4 is required to elicit cankers in sweet orange leaves and that depletion of CsMAF1 in X. citri-infected tissues correlates with the development of hyperplastic lesions and the presence of PthA4. Considering that CsMAF1 and CsCYP function as canker suppressors in sweet orange, our data indicate that TAL effectors from X. citri target negative regulators of RNA Pol II and Pol III to coordinately increase the transcription of host genes involved in ribosome biogenesis and cell proliferation.

Increased resistance against citrus canker mediated by a citrus mitogen-activated protein kinase.

Mitogen-activated protein kinases (MAPK) play crucial roles in plant immunity. We previously identified a citrus MAPK (CsMAPK1) as a differentially expressed protein in response to infection by Xanthomonas aurantifolii, a bacterium that causes citrus canker in Mexican lime but a hypersensitive reaction in sweet oranges. Here, we confirm that, in sweet orange, CsMAPK1 is rapidly and preferentially induced by X. aurantifolii relative to Xanthomonas citri. To investigate the role of CsMAPK1 in citrus canker resistance, we expressed CsMAPK1 in citrus plants under the control of the PR5 gene promoter, which is induced by Xanthomonas infection and wounding. Increased expression of CsMAPK1 correlated with a reduction in canker symptoms and a decrease in bacterial growth. Canker lesions in plants with higher CsMAPK1 levels were smaller and showed fewer signs of epidermal rupture. Transgenic plants also revealed higher transcript levels of defense-related genes and a significant accumulation of hydrogen peroxide in response to wounding or X. citri infection. Accordingly, nontransgenic sweet orange leaves accumulate both CsMAPK1 and hydrogen peroxide in response to X. aurantifolii but not X. citri infection. These data, thus, indicate that CsMAPK1 functions in the citrus canker defense response by inducing defense gene expression and reactive oxygen species accumulation during infection.

Secondary Metabolites of Pseudomonas aeruginosa LV Strain Decrease Asian Soybean Rust Severity in Experimentally Infected Plants.

Asian Soybean Rust (ASR), a disease caused by Phakopsora pachyrhizi, causing yield losses up to 90%. The control is based on the fungicides which may generate resistant fungi. The activation of the plant defense system, should help on ASR control. In this study, secondary metabolites of Pseudomonas aeruginosa LV strain were applied on spore germination and the expression of defense genes in infected soybean plants. The F4A fraction and the pure metabolites were used. In vitro, 10 µg mL-1 of F4A reduced spore germination by 54%, while 100 µg mL-1 completely inhibited. Overexpression of phenylalanine ammonia lyase (PAL), O-methyltransferase (OMT) and pathogenesis related protein-2 (PR-2; glucanases) defense-related genes were detected 24 and 72 h after soybean sprouts were sprayed with an organocopper antimicrobial compound (OAC). Under greenhouse conditions, the best control was observed in plants treated with 60 µg mL-1 of PCA, which reduced ASR severity and lesion frequency by 75% and 43%, respectively. Plants sprayed with 2 and 20 µg mL-1 of F4A also decreased severity (41%) and lesion frequency (32%). The significant reduction in spore germination ASR in plant suggested that the strain of these metabolites are effective against P. pachyrhizi, and they can be used for ASR control.

Additive roles of PthAs in bacterial growth and pathogenicity associated with nucleotide polymorphisms in effector-binding elements of citrus canker susceptibility genes.

Citrus canker, caused by Xanthomonas citri, affects most commercial citrus varieties. All X. citri strains possess at least one transcription activator-like effector of the PthA family that activates host disease susceptibility (S) genes. The X. citri strain 306 encodes four PthA effectors; nevertheless, only PthA4 is known to elicit cankers on citrus. As none of the PthAs act as avirulence factors on citrus, we hypothesized that PthAs 1-3 might also contribute to pathogenicity on certain hosts. Here, we show that, although PthA4 is indispensable for canker formation in six Brazilian citrus varieties, PthAs 1 and 3 contribute to canker development in 'Pera' sweet orange, but not in 'Tahiti' lemon. Deletions in two or more pthA genes reduce bacterial growth in planta more pronouncedly than single deletions, suggesting an additive role of PthAs in pathogenicity and bacterial fitness. The contribution of PthAs 1 and 3 in canker formation in 'Pera' plants does not correlate with the activation of the canker S gene, LOB1 (LATERAL ORGAN BOUNDARIES 1), but with the induction of other PthA targets, including LOB2 and citrus dioxygenase (DIOX). LOB1, LOB2 and DIOX show differential PthA-dependent expression between 'Pera' and 'Tahiti' plants that appears to be associated with nucleotide polymorphisms found at or near PthA-binding sites. We also present evidence that LOB1 activation alone is not sufficient to elicit cankers on citrus, and that DIOX acts as a canker S gene in 'Pera', but not 'Tahiti', plants. Our results suggest that the activation of multiple S genes, such as LOB1 and DIOX, is necessary for full canker development.